Tetra-O-substituted butane-bridge modified NDGA derivatives, their synthesis and pharmaceutical use

ABSTRACT

The present invention relates to nordihydroguaiaretic acid derivative compounds, namely, butane bridge modified nordihydroguaiaretic acid (NDGA) compounds and butane bridge modified tetra-O-substituted NDGA compounds, pharmaceutical compositions containing them, methods of making them, and methods of using them and kits including them for the treatment of diseases and disorders, in particular, diseases resulting from or associated with a virus infection, such as HIV infection, HPV infection, or HSV infection, an inflammatory disease, such as various types of arthritis and inflammatory bowel diseases, metabolic diseases, such as diabetes and hypertension, or a proliferative disease, such as diverse types of cancers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application 13/561,517, filed onJul. 30, 2012, now U.S. Pat. No. 8,691,845, which is a divisionalapplication of U.S. patent application Ser. No. 12/443,926, filed May18, 2009, which is an U.S. National Phase application under 35 U.S.C.§371 of International Patent Application No. PCT/US2007/080231, filedOct. 2, 2007, which claims the benefit of U.S. Provisional ApplicationNo. 60/827,783, filed Oct. 2, 2006; the contents of which areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

The present invention relates to nordihydroguaiaretic acid derivatives,methods of making them and methods of using them for treating viralinfections, inflammatory diseases, metabolic diseases, vascular(including cardiovascular) diseases and proliferative diseases, such ascancer.

Nordihydroguaiaretic acid (NDGA, Formula I) has the following chemicalstructure, in which there are two catechol groups, and a2,3-dimethylbutane bridge. The butane bridge links two catechol moietiesthrough a 4 position. NDGA is a natural compound that can be isolatedfrom the resin of the leaves of Larrea tridentate, a desert plantindigenous to the southwestern United States and Mexico. It has ameso-form conformation of (2S,3R), which is the symmetric structure, andis not optical active.

Research on NDGA and its derivatives has been attracting increasinginterest recently. A large number of NDGA derivatives have beenreported, and could be classified as the following:

Type 1: ether bonded NDGA, the most common NDGA derivatives, in which asubstituted group is chemically bonded to one or more of the hydroxygroups of the catechol moieties.

Type 2: ester bonded NDGA derivatives, in which a substituted group iscovalently bonded to one or more of the hydroxy groups of the catecholmoieties.

Type 3: end-ring NDGA derivatives, in which two hydroxy groups at thecatechol moieties were linked together to form 5-6 member rings throughether or carbonate bonds.

Type 4: di-substituted NDGA derivatives, in which one hydroxy group ofthe catechol is methylated, the other one is covalently bonded to asubstituted group.

Type 5: phenyl ring modifications, in which the substituted groups arechemically linked to the phenyl ring.

Type 6: Butane bridge modifications, in which two methyl groups in thebridge were removed or modified by substituted groups.

NDGA and its synthetic derivatives have numerous characteristics. Beinga lipoxygenases inhibitor, NDGA can induce cystic nephropathy in therat.¹ In addition, it shows various bioactivities, including inhibitionof protein kinase C,² induction of apoptosis,³ alterations of thecellular membrane,⁴ elevation of cellular Ca²⁺ level⁵ and activation ofCa²⁺ channels in smooth muscle cells,⁶ breakdown of pre-formedAlzheimer's beta-amyloid fibrils in vitro,⁷ anti-oxidation,⁸ etc. Thisnatural product NDGA is used commercially as a food additive to preservefats and butter in some parts of the world. Recently, the derivatives ofthe plant lignan NDGA have been used to block viral replication throughthe inhibition of viral transcription.⁹⁻¹⁶ These compounds can inhibitproduction of human immunodeficiency virus (HIV),⁹⁻¹³ herpes simlexvirus (HSV),^(14, 15) and human papillomavirus (HPV) transcripts¹⁶ bydeactivation of their Sp1-dependent promoters. Moreover,(tetra-O-methyl)nordihydroguaiaretic acid (M₄N, Formula II,terameprocol) can function as an anti-HIV proviral transcriptioninhibitor and causes growth arrest of a variety of transformed human andmouse cells in culture and in mice.¹⁷⁻¹⁹ Compound M₄N is currently inclinical trials against human cancers.

While M₄N (Formula II) is a strikingly effective and non-toxicanticancer agent, M₄N and several other methylated NDGAs, all show poorwater solubility which somewhat limit their application for certain drugaction studies. To circumvent this problem, a water soluble derivativeof NDGA, (tetra-O-dimethylglycyl)nordihydroguaiaretic acid (G₄N, FormulaIII) has been designed and synthesized.^(11, 18)

G₄N is a very effective mutation-insensitive inhibitor to HIV-1, HSV-1and HSV-2.^(17, 22) However, it is somewhat unstable and has arelatively short half-life in aqueous solution, reportedly due to theester bonds connecting the dimethyl glycine moieties on the NDGA mainskeleton.¹⁸

Therefore, there is a need for NDGA derivatives, some with improvedwater solubility, as well as good stability, both as water solublecompound and as hydrophobic compounds having the desired pharmaceuticaleffects. The inventors have developed new derivatives of NDGA that havethese advantages and will be useful in therapeutic compositions andtreatment methods.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to nordihydroguaiaretic acid derivativecompounds, pharmaceutical compositions, methods of making them, andmethods of using them and kits including them for the treatment ofdiseases and disorders, in particular, diseases resulting from orassociated with a virus infection, an inflammatory disease, a metabolicdisease, a vascular disease or a proliferative disease.

The present invention is based on research stemming from the inventors'realization that 1,4-bis(catechol-4-yl)-butane (Formula IV), adouble-demethylated NDGA, showed 10 times more potency than NDGA as aproliferative inhibitor of H-69 small cell lung cancer cells (MaDonal,R. W.; Bunjobpon, W; Liu, T.; Fessler, S.; Pardo, O. E.; Freer, I. K.A.; Glaser, M.; Seckl, M. J.; Robins, D. J.; Anti-cancer Drug Design,2001, 16(6), 261-270). The inventors have invented a novel NDGAderivative based on butane bridge modification designated BB-N, where BBsignifies a butane bridge modification, and N signifies NDGA in thefollowing Formula V:

One aspect of the present invention relates to a butane bridge-modifiednordihydroguaiaretic acid derivative compound designated “BB-N”, havingthe foregoing general structure (Formula V), as well as itspharmaceutically acceptable salts.

In the compound of Formula V, each X is selected from the groupconsisting of —CHO, —CN, —CH₂Cl, —CH₂Br and —CH₂F. However, where theBB-N derivative is used as an intermediate to make the “BB-Sb₄N”derivative disclosed below, X may also be H or —CH₂CH₃, in which case,the designation “X” will be replaced in the appropriate formulas by “Z”.

Another aspect of the present invention relates to a butane bridgemodified tetra-O-substituted nordihydroguaiaretic acid derivativecompound designated “BB-Sb₄N”, having the following general structure(Formula VI), as well as its pharmaceutically acceptable salts:

In the designation “BB-Sb₄N”, “BB” signifies the butane bridgemodification aspect of the NDGA base component N and “Sb₄N” signifiesthe tetra-O-substituted nordihydroguaiaretic acid aspect of the bridgemodified tetra-O-substituted nordihydroguaiaretic acid derivativecompound of the present invention. The compound contains two catecholunits, a butane bridge, and a four groups Y, each substituted for H inthe NDGA hydroxyl groups (sometimes referred to as the “substitutedgroup Y”) designated by “Sb₄” in “Sb₄N.”

The butane bridge links the two catechol units at the 1,4-positionsthrough the 4-position at the phenyl rings of the catechol units. Thesubstituents (Z) at the 2,3-positions of the butane bridge are selectedfrom the group consisting of H, —CHO, —CN, —CH₂CH₃, —CH₂Cl, —CH₂Br and—CH₂F.

Y is selected from the group consisting of:

-A-R;

—(CH₂)_(x)Hal, where x is an integer of 1 to 10, and Hal is a halogenatom, namely any of chlorine, fluorine, bromine or iodine;

—(CH₂CH₂O)_(y)H, where y is an integer of 1 to 10; and

a carbamate-bonded group selected from the group consisting of:

where n is an integer of 1 to 6, T₁ is a saturated linear hydrocarbonchain of 2-6 carbons and optionally 1-3 halogen atoms, T₂ is a 5- to7-member ring optionally containing 0-3 double bonds and optionallycontaining 1-3 atoms of any of O, N and S, and T₃ is methyl or ethyl.

When Y is -A-R, R is an end group and A is a linear saturatedhydrocarbon side chain with optional heteroatoms that is bonded at oneend to the respective hydroxy residue O groups by an ether bond or acarbamate bond and at the other end to a carbon or a heteroatom in theend group R.

The side chain A is selected from the group consisting of a C₂-C₁₆linear saturated hydrocarbon chain, optionally with 1-5 heteroatomsselected from the group consisting of O, N and S, bonded to therespective hydroxy residue O groups of NGDA through an ether bond; and1-5 units of a polyethylene glycol (PEG) chain.

The end group R is selected from the group consisting of:

a 5- to 7-member carbocyclic ring selected from the group consisting ofa fully saturated ring with 1 to 3 N, O or S heteroatoms; a ringcontaining 1 to 3 double bonds for a 6- or 7-member ring and 1 to 2double bonds for a 5-member ring, with 1 to 3 N, O or S heteroatoms forthe 5 to 7 member ring; a ring containing a carbamate bond, a urea bond,a carbonate bond or an amide bond; and

a water soluble group selected from the group consisting of an alkalimetal salt of sulfonic acid; an alkali metal salt of phosphonic acid; apharmaceutically acceptable salt; a sugar and a polyhydroxy group.

Another aspect of the invention is a composition comprising the BB-Ncompound or the BB-Sb₄N compound and a pharmaceutically acceptablecarrier, optionally with other pharmaceutically acceptable excipients.

Still another aspect of the invention is a method of making the BB-Ncompound or the BB-Sb₄N compound as set forth hereinafter.

Another aspect of the invention is a method of administering to asubject, an amount of either of the BB-N compound or the BB-Sb₄Ncompound alone or as part of a pharmaceutical composition effectiveprophylactically or for treating a viral infection.

Yet another aspect of the invention is a method of administering to asubject an amount of either the BB-N compound or the BB-Sb₄N compoundalone or as part of a pharmaceutical composition effectiveprophylactically or for treating a proliferative disease.

Another aspect of the invention is a method of administering to asubject an amount of either the BB-N compound or the BB-Sb₄N compoundalone or as part of a pharmaceutical composition effectiveprophylactically or for treating an inflammatory disease.

Yet another aspect of the invention is a method of administering to asubject an amount of either the BB-N compound or the BB-Sb₄N compoundalone or as part of a pharmaceutical composition effectiveprophylactically or for treating a metabolic disease.

A further aspect of the invention is a method of administering to asubject an amount of either the BB-N compound or the BB-Sb₄N compoundalone or as part of a pharmaceutical composition effectiveprophylactically or for treating a vascular disease.

Still another aspect of the present invention is a kit comprising apharmaceutical composition comprising either the BB-N compound or theBB-Sb₄N compound and instructions for its use prophylactically or fortreating a viral infection, a proliferative disease, an inflammatorydisease, a metabolic disease or a vascular disease.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention relates to nordihydroguaiareticacid derivative compounds, pharmaceutical compositions containing them,methods of making them, and methods of using them and kits includingthem for the treatment of diseases and disorders, in particular, viralinfections, such as, for example and without limitation, infectionscaused by human immunodeficiency virus (HIV), human papillomaviruses(HPV)(all subtypes), herpes simplex virus 1 and 2 (HSV-1 and HSV-2),Varicella Zoster virus, cytomegalovirus, Epstein Barr virus, poxviruses(smallpox, cowpox, monkeypox, vaccinia), orthohepadnavirus, JCvirus, and BK virus; inflammatory diseases, such as, for example andwithout limitation, various types of arthritis and inflammatory boweldiseases; metabolic diseases, such as, for example and withoutlimitation, diabetes; vascular diseases, such as for examplehypertension, cardiovascular diseases and macular degeneration; andproliferative diseases such as various types of cancers.

The present invention is based on considerations including experiencewith agents used for treating cancer and viruses, including HIV;derivatives having a chemical structure related to NDGA; where suchderivatives have more potency, better PD/PK profile and less or no sideeffects versus (tetra-O-methyl)nordihydroguaiaretic acid (M₄N)(terameprocol), at least some formulations of which are orallybioavailable. The NDGA derivatives of the present invention are in themeso form without any possible mixtures of their enantiomers, which willmake the chemical and biological characterization easier. Thesubstituent functional groups for the modifications of the NDGA parentcompound are selected from among the most common chemical groups usedfor successful drug molecule modifications. They are readily able to besynthesized and readily formulated with reasonable aqueous solubility,in that in the HCl or other salt form or in free base, they haveconsiderable aqueous solubility. Other of the NDGA derivatives of thepresent invention are hydrophobic. The NDGA derivatives of the presentinvention have good stability, whether they are water soluble compoundor hydrophobic compounds. The derivatives may be scaled-up readily forcommercial production.

The NDGA derivatives of the present invention were developed based onthe fact that NDGA is natural compound with a broad range of biologicalactivities. NDGA has a lot of side effects, which are overcome by thederivatives of the invention. Butane-bridge modified NDGA (BB-N)derivatives, such as 1,4-bis(catechol-4-yl)-butane, possess betterbiological activities than NDGA. The derivatives have improvedbiological activities. The research leading to the development of thepresent invention has also shown that hydroxy group modification of NDGAderivatives, such as M₄N, prevents tumor cell replication andselectively induces tumor cell death (apoptosis). This is achieved bypreventing Sp1-regulated production of cdc2 (p34) and survivin. Survivinis an inhibitor of apoptosis protein (IAP) over-expressed inpre-cancerous and cancerous cells, and rarely found in healthy adultcells. M₄N also prevents proliferation of human immunodeficiency virus(HIV), herpes simplex virus (HSV), and human papilloma virus (HPV). Thisis achieved through the deactivation of viral Sp1-dependent promotersthat are essential for viral propagation. BB-Sb₄N derivatives of thepresent invention will remarkably improve their activities to preventSp1-regulated production of cdc2 and survivin by using a suitablefunctional group to modify the hydroxyl group of NDGA.

Definitions

A “buffer” suitable for use herein includes any buffer conventional inthe art, such as, for example, Tris, phosphate, imidazole, andbicarbonate.

A “cyclodextrin” as used herein means an unmodified cyclodextrin or amodified cyclodextrin, and includes with out limitation α-cyclodextrin,β-cyclodextrin, γ-cyclodextrin and any modified cyclodextrins containingmodifications thereto, such as hydoxypropyl-β-cyclodextrin (“HP-β-CD”)or sulfobutyl ether β-cyclodextrin (“SBE-β-CD”). Cyclodextrin typicallyhas 6 (α-cyclodextrin), 7 (β-cyclodextrin), and 8 (γ-cyclodextrin)sugars, up to three substitutions per sugar, and 0 to 24 primarysubstitutions are therefore possible (primary substitutions are definedas substitutions connected directly to the cyclodextrin ring). Themodified or unmodified cyclodextrins used in the present invention mayhave any appropriate number and location of primary substitutions orother modifications.

An “NDGA derivative” of the present invention as used herein means aderivative of NDGA designated as “BB-N” or “BB-Sb₄N” hereinafter.

A “pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any conventional type. A “pharmaceuticallyacceptable carrier” is non-toxic to recipients at the dosages andconcentrations employed, and is compatible with other ingredients of theformulation. For example, the carrier for a formulation containing thepresent catecholic butane or NDGA derivatives preferably does notinclude oxidizing agents and other compounds that are known to bedeleterious to such derivatives. Suitable carriers include, but are notlimited to, water, dextrose, glycerol, saline, ethanol, buffer, dimethylsulfoxide, Cremaphor EL, and combinations thereof. The carrier maycontain additional agents such as wetting or emulsifying agents, or pHbuffering agents. Other materials such as anti-oxidants, humectants,viscosity stabilizers, and similar agents may be added as necessary.

A “pharmaceutically acceptable salt” as used herein includes the acidaddition salts (formed with the free amino groups of the polypeptide)and which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,mandelic and oxalic acids. Salts formed with the free carboxyl groupsmay also be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol, andhistidine. Non-limiting examples of pharmaceutically acceptable salts ofthe NDGA derivatives of the present invention include, for instance, thefollowing general formula of salts:[Sb₄(BB-N)]·k[acid],

where BB-N is butane-bridge modified NDGA, Sb is a substituted group asdescribed in Table 1 and 2, k is an integer or non integer number, andacid is organic or inorganic acid, as exemplified in the followingnon-limiting Table A:

TABLE A Sb Acid k Containing HCl, HBr, HNO₃, MeSO₃H, H₂SO₄, asparticacid, 1-4 one basic citric acid, benzenesulfonic acid, camphoric acid,nitrogen camphorsulfonic acid, ethanesulfonic acid, 2-hydroxy- atomethansulfonic acid, formic acid, fumaric acid, galactaric acid,D-gluconic acid, glycolic acid, hippuric acid, L- lactic acid, maleicacid, malic acid, malonic acid, nicotinic acid, palmitic acid, pamoicacid, phosphoric acid, salicylic acid, succinic acid, tartaric acid, p-toluenesulfonic acid. Containing HCl, HBr, HNO₃, MeSO₃H, H₂SO₄, asparticacid, 1-8 two basic citric acid, benzenesulfonic acid, camphoric acid,nitrogen camphorsulfonic acid, ethanesulfonic acid, 2-hydroxy- atomsethansulfonic acid, formic acid, fumaric acid, galactaric acid,D-gluconic acid, glycolic acid, hippuric acid, L- lactic acid, maleicacid, malic acid, malonic acid, nicotinic acid, palmitic acid, pamoicacid, phosphoric acid, salicylic acid, succinic acid, tartaric acid, p-toluenesulfonic acid.

The term “pharmaceutically acceptable excipient” as used herein includesvehicles, adjuvants, or diluents or other auxiliary substances, such asthose conventional in the art, which are readily available to thepublic. For example, pharmaceutically acceptable auxiliary substancesinclude pH adjusting and buffering agents, tonicity adjusting agents,stabilizers, wetting agents and the like.

A “ring” unless otherwise specified, as used herein such as in the terms“5-member ring,” “6-member ring” and “7-member ring,” refers to acarbocyclic ring with any indicated heteroatoms.

The terms “subject,” “host,” and “patient,” are used interchangeablyherein to refer to an animal being treated with the presentcompositions, including, but not limited to, simians, humans, felines,canines, equines, bovines, porcines, ovines, caprines, mammalian farmanimals, mammalian sport animals, and mammalian pets.

A “substantially purified” compound in reference to the NDGA derivativesherein is one that is substantially free of compounds that are not theNDGA derivatives of the present invention (hereafter, “non-NDGAderivative materials”). By substantially free is meant at least 50%,preferably at least 70%, more preferably at least 80%, and even morepreferably at least 90% free of non-NDGA derivative materials.

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a condition or disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a condition ordisease and/or adverse affect attributable to the condition or disease.“Treatment,” thus, for example, covers any treatment of a condition ordisease in an animal, preferably in a mammal, and more preferably in ahuman, and includes: (a) preventing the condition or disease fromoccurring in a subject which may be predisposed to the condition ordisease but has not yet been diagnosed as having it; (b) inhibiting thecondition or disease, such as, arresting its development; and (c)relieving, alleviating or ameliorating the condition or disease, suchas, for example, causing regression of the condition or disease.

This invention is described by way of example only and is not to beinterpreted in any way as limiting the invention. Thus, this inventionis not limited to particular embodiments described, as such may, ofcourse, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to be limiting, since the scope of the present inventionwill be limited only by the appended claims in a non-provisionalapplication based on this provisional application.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

It must be noted that as used herein, the singular forms “a”, “an”, and“the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a derivative” includes aplurality of such derivatives and reference to “the NDGA derivative”includes reference to one or more NDGA derivatives and equivalentsthereof known to those skilled in the art.

All publications mentioned herein, including patents, patentapplications, and journal articles are incorporated herein by referencein their entireties including the references cited therein, which arealso incorporated herein by reference. The publications discussed hereinare provided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention. Further, the dates of publication providedmay be different from the actual publication dates which may need to beindependently confirmed.

As noted above, one aspect of the present invention relates to a butanebridge-modified nordihydroguaiaretic acid derivative compound designated“BB-N”, having the general structure (Formula V), as well as itspharmaceutically acceptable salts.

In the compound of Formula V, each X is selected from the groupconsisting of —CHO, —CN, —CH₂Cl, —CH₂Br and —CH₂F. However, as notedabove, where the BB-N derivative is used as an intermediate to make the“BB-Sb₄N” derivative disclosed below, X may also be H or —CH₂CH₃, inwhich case, the designation “X” will be replaced in the appropriateformulas by “Z”.

Another aspect of the present invention relates to a butane bridgemodified tetra-O-substituted nordihydroguaiaretic acid derivativecompound designated “BB-Sb₄N”, having the following general structure(Formula VI), as well as its pharmaceutically acceptable salts:

As also noted above, Y is selected from the group consisting of:

-A-R;

—(CH₂)_(x)Hal, where x is an integer of 1 to 10, and Hal is a halogenatom, namely any of chlorine, fluorine, bromine or iodine;

—(CH₂CH₂O)_(y)H, where y is an integer of 1 to 10; and

a carbamate-bonded group selected from the group consisting of:

where n is an integer of 1 to 6, T₁ is a saturated linear hydrocarbonchain of 2-6 carbons and optionally 1-3 halogen atoms, T₂ is a 5- to7-member ring optionally containing 0-3 double bonds and optionallycontaining 1-3 atoms of any of O, N and S, and T₃ is methyl or ethyl.

Where Y is —(CH₂)_(x)Hal, x is preferably 1 to 3, and Hal is chlorine orfluorine; more preferably, in this instance, Y is —(CH₂)₂F, for example.

Where Y is —(CH₂CH₂O)_(y)H, y is preferably 1 to 3, and more preferably,for example, in this instance, Y is —(CH₂)₂OH (when y is 1); or—(CH₂)₂—O—(CH₂)₂—OH (when y is 2).

With the foregoing definitions of Y, the butane bridge substituents Z,and their side chains A and end groups R of the substituted Y groups ofthe BB-Sb₄N derivative of NDGA may be selected from those set forth inthe Brief Summary of the Invention above, and are also set forth intabular form in the following Table 1.

TABLE 1 Z group —CHO, —CN, —CH₂CH₃, —CH₂Cl, —CH₂Br or —CH₂F Side chain AC₂-C₁₆ linear saturated hydrocarbon chain optionally with 1-5 N, O or Sheteroatoms, and the chain is bonded to the respective hydroxy groupsresidue O groups of the phenyl moieties through an ether bond 1-5 unitsof polyethylene glycol (PEG) chain R is a 7 fully saturated 7-memberring with 1 to 3 N, O or S member ring heteroatoms 7-member ringcontaining 1 to 3 double bonds with 1 to 3 N, O or S heteroatoms7-member ring containing a carbamate bond, a urea bond, a carbonate bondor an amide bond R is a 6 fully saturated 6-member ring with 1 to 3 N, Oor S member ring heteroatoms 6-member ring containing 1 to 3 doublebonds with 1 to 3 N, O or S heteroatoms 6-member ring containing acarbamate bond, a urea bond, a carbonate bond or an amide bond R is a 5fully saturated 5-member ring with 1 to 3 N, O or S member ringheteroatoms 5-member ring containing 1 to 2 double bonds with 1 to 3 N,O or S heteroatoms 5-member ring containing a carbamate bond, a ureabond, a carbonate bond or an amide bond R is a water an alkali metalsalt of sulfonic acid soluble group an alkali metal salt of phosphonicacid a pharmaceutically acceptable salt, such as shown in Table A asugar a polyhydroxy group

Non-limiting examples of a suitable side chain A are C₂-C₄ linear chain,such as ethylene, propylene or butylene, bonded at one end to therespective hydroxy residue O groups of NDGA through an ether bond; C₂-C₄linear chain, such as ethylene, propylene or butylene, with an O or Nheteroatom, and the chain is bonded to the respective hydroxy residue Ogroups of NGDA through an ether bond; 1-3 units of polyethylene glycol(PEG) chain; or a carbamate bond. The side chain is bonded at the otherend to a carbon or heteroatom of the end group R.

Nonlimiting examples of suitable Y and R end groups are set forth in thefollowing Table 2:

TABLE 2 R is 5-7 member carbocyclic ring containing 1-3 N, O or Sheteroatoms

R is polyhydroxy, sugar, or other water soluble group, where m is aninteger of 2 to 6

—(CH₂)_(m)SO₃Na —(CH₂)mPO₃Na —(CH₂)mSO₂NH₂ Sugar

Methods to synthesize butane bridge modified NDGA (BB-N) and the butanebridge modified tetra-O-substituted nordihydroguaiaretic acid derivativecompound designated (BB-Sb₄N) are as set forth below.

Several methods to synthesize 1,4-bis(catechol-4-yl)-butane, one ofstarting materials, were reported in the literature (such as by multiplestep synthesis, see Anticancer Drug Design, 2001, 16, 261-270; and byhomo-coupling the corresponding boronic acid, see Tetrahedron Lett,2002, 43, 8149-8151). However, those methods gave low yields of theexpected product, and some starting materials are not readily available.

To overcome the deficiencies of the prior synthesis methods, theinventors developed a new method to synthesize the expected1,4-bis(catechol-4-yl)-butane, which is obtained in remarkably highyield by a simplified procedure.

McMurry coupling an aldehyde to give a mixture of cis- and trans- formcompounds, which is reduced to give the expected tetra-O-methyl-BB-N asa precursor for the BB-N derivatives of Formula V:

Other butane bridge substituted 1,4-bis(catechol-4-yl)butanes can besynthesized as follows:

The starting 2,3-bis(hydroxymethyl)-1,4-bis(3,4-dimethoxyphenyl)-butanecould be synthesized according to literature (JACS, 1957, 79, 3823-3827;Tetrahedron: Ass. 1998, 9, 2827-2831; Tetrahedron 1996, 52(39),12799-12814; Tetrahedron Ass. 1995, 6(4), 843-844.)

Methods to synthesize the ether bonded butane bridge modifiedtetra-O-substituted NDGA derivatives BB-Sb₄N (Formula VI) from thebutane bridge modified NDGA derivatives BB-N(Formula V, where the Xgroups are replaced by the Z groups as explained above) are set forthbelow.

General method 1: reaction of alkyl halide with butane bridge modifiedNDGA (BB-N) under basic catalytic conditions:

General method 2: reaction of toluenesulfonic acid activated alcoholwith butane bridge modified NDGA (BB-N):

Methods to synthesize carbamate bonded butane bridge modifiedtetra-O-substituted NDGA derivatives (BB-Sb₄N):

General method 1: reaction of an isocyanate compound with BB-N

General method 2: BB-N is reacted with an activated amino compound

Details of the preparation of specific NDGA derivative compounds BB-Nand BB-S₄N according to the present invention will be set forth below inthe Examples section.

The present NDGA derivatives in a suitable formulation, preferably butnot exclusively as the active ingredient or as one of two or more activeingredients in a pharmaceutical composition with a pharmaceuticallyacceptable carrier or excipient where appropriate, can be safelyadministered to a subject in need of such treatment by intranasaldelivery, by inhalation, intravenously such as by infusion or byinjection into the central vein for example, intra-arterially (with orwithout occlusion), intraperitoneally, interstitially, subcutaneously,transdermally, intradermally, intraocularly, intramuscularly, topically,intracranially, intraventricularly, orally, or buccally, or byimplantation.

Moreover, the NDGA derivatives can be safely administered to a subjectin need of such treatment in solution, suspension, semisolid or solidforms as appropriate, or in liposomal formulations, nanoparticleformulations, or micellar formulations for administration via one ormore routes mentioned above.

Furthermore, the NDGA derivatives in liposomal formulations,nanoparticles formulations, or micellar formulations can be embedded ina biodegradable polymer formulation and safely administered, such as bysubcutaneous implantation.

Compositions for administration herein may, be in any suitable form,such as and without limitation, a solution, suspension, tablet, pill,capsule, sustained release formulation or powder, a liquid that iseither hydrophilic or hydrophobic, a powder such as one resulting fromlyophilization, an aerosol, an aqueous or water-soluble composition, ahydrophobic composition, a liposomal composition, a micellar compositionsuch as that based on Tween® 80 or diblock copolymers, a nanoparticlecomposition, a polymer composition, a cyclodextrin complex composition,an emulsion, or as lipid based nanoparticles termed “lipocores.”

The present invention further encompasses compositions, includingpharmaceutical compositions, comprising the NDGA derivatives andpharmaceutically acceptable carriers or excipients. These compositionsmay include a buffer, which is selected according to the desired use ofthe NDGA derivatives, and may also include other substances appropriatefor the intended use. Those skilled in the art can readily select anappropriate buffer, a wide variety of which are known in the art,suitable for an intended use, in view of the present disclosure. In someinstances, the composition can comprise a pharmaceutically acceptableexcipient, a variety of which are known in the art. Pharmaceuticallyacceptable excipients suitable for use herein are described in a varietyof publications, including, for example, Gennaro (Gennaro, A.,Remington: The Science and Practice of Pharmacy, 19th edition,Lippincott, Williams, & Wilkins, (1995)); Ansel, et al. (Ansel, H. C. etal., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7^(th)edition, Lippincott, Williams, & Wilkins (1999)); and Kibbe (Kibbe, A.H., Handbook of Pharmaceutical Excipients, 3^(rd) edition Amer.Pharmaceutical Assoc.).

The compositions herein are formulated in accordance to the mode ofpotential administration. Thus, if the composition is intended to beadministered intranasally or by inhalation, for example, the compositionmay be a converted to a powder or aerosol form, as conventional in theart, for such purposes. Other formulations, such as for oral orparenteral delivery, are also used as conventional in the art.

Compositions or formulations suitable for oral or injectable deliveryadditionally includes a pharmaceutical composition containing acatecholic butane for treatment of the indicated diseases where thecomposition is formulated with a pharmaceutically acceptable carrier andother optional excipients, wherein the carrier comprises at least one ofa solubilizing agent and an excipient selected from the group consistingof: (a) a water-soluble organic solvent; (b) a cyclodextrin (including amodified cyclodextrin); (c) an ionic, non-ionic or amphipathicsurfactant, (d) a modified cellulose; (e) a water-insoluble lipid; and acombination of any of the carriers (a)-(e).

The water-soluble organic solvent may be preferably, but notnecessarily, other than dimethyl sulfoxide. Non-limiting exemplarywater-soluble organic insolvents include polyethylene glycol (“PEG”),for example, PEG 300, PEG 400 or PEG 400 monolaurate, propylene glycol(“PG”), polyvinyl pyrrolidone (“PVP”), ethanol, benzyl alcohol ordimethylacetamide.

The cyclodextrin or modified cyclodextrin may be, without limitation,α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, HP-β-CD or SBE-β-CD.

The ionic, non-ionic or amphipathic surfactant may include, for examplewithout limitation, a surfactant such as polyoxyethylene sorbitanmonolaurate (also known as polysorbate), which is a non-ionicsurfactant, for example, polysorbate 20 and polysorbate 80, commerciallyavailable as Tween® 20 or Tween® 80, d-alpha-tocopheryl polyethyleneglycol 1000 succinate (“TPGS”), glycerol monooleate (also known asglyceryl monooleate), an esterified fatty acid or a reaction productbetween ethylene oxide and castor oil in a molar ratio of 35:1,commercially available as Cremophor® EL. Preferably, for certainembodiments, when the surfactant is a non-ionic surfactant, thenon-ionic surfactant is present in the absence of xanthan gum.

Non-limiting examples of a modified cellulose include ethyl cellulose(“EC”), hydroxylpropyl methylcellulose (“HPMC”), methylcellulose (“MC”)or carboxy methylcellulose (“CMC”). In one embodiment of the invention,the catecholic butane may be solubilized in modified celluloses that canbe diluted in ethanol (“EtOH”) prior to use.

The water-insoluble lipids include, for example, an oil or oils, such ascastor oil, sesame oil or peppermint oil, a wax or waxes, such asbeeswax or carnuba wax, and mixed fat emulsion compositions such asIntralipid® (Pharmacia & Upjohn, now Pfizer), used as per themanufacturer's recommendation. For example, adult dosage is recommendedto be not exceeding 2 g of fat/kg body weight/day (20 mL, 10 mL and 6.7mL/kg of Intralipid® 10%, 20% and 30%, respectively). Intralipid® 10% isbelieved to contain in 1,000 mL: purified soybean oil 100 g, purifiedegg phospholipids 12 g, glycerol anhydrous 22 g, water for injectionq.s. ad 1,000 mL. pH is adjusted with sodium hydroxide to pHapproximately 8. Intralipid® 20% contains in 1,000 mL: purified soybeanoil 200 g, purified egg phospholipids 12 g, glycerol anhydrous 22 g,water for injection q.s. ad 1,000 mL. pH is adjusted with sodiumhydroxide to pH approximately 8. Intralipid® 30% contains in 1,000 mL:purified soybean oil 300 g, purified egg phospholipids 12 g, glycerolanhydrous 16.7 g, water for injection q.s. ad 1,000 mL. pH is adjustedwith sodium hydroxide to pH approximately 7.5. These Intralipid®products are stored at controlled room temperature below 25° C. andshould not be frozen.

In one embodiment of the invention, the NDGA derivative is dissolved ordissolved and diluted in different carriers to form a liquid compositionfor oral administration into animals, including humans. For example, inone aspect of this embodiment, the NDGA derivative is dissolved in awater-soluble organic solvent such as a PEG 300, PEG 400 or a PEG 400monolaurate (the “PEG compounds”) or in PG. In another embodiment, theNDGA derivative is dissolved in a modified cyclodextrin, such as HP-β-CDor SBE-β-CD. In yet another embodiment, the present NDGA derivative issolubilized and/or diluted in a combination formulation containing a PEGcompound and HP-β-CD. In a further embodiment, the NDGA derivativeherein is dissolved in a modified cellulose such as HPMC, CMC or EC. Inyet another embodiment, the NDGA derivative herein is dissolved inanother combination formulation containing both a modified cyclodextrinand modified cellulose, such as, for example, HP-β-CD and HPMC orHP-β-CD and CMC.

In yet another embodiment, the NDGA derivative is dissolved in ionic,non-ionic or amphipathic surfactants such as Tween® 20, Tween® 80, TPGSor an esterified fatty acid. For example, the present compounds can bedissolved in TPGS alone, or Tween® 20 alone, or in combinations such asTPGS and PEG 400, or Tween® 20 and PEG 400.

In a further embodiment, the present NDGA derivative is dissolved in awater-insoluble lipid such as a wax, fat emulsion, for exampleIntralipid®, or oil. For example, the present compounds can be dissolvedin peppermint oil alone, or in combinations of peppermint oil withTween® 20 and PEG 400, or peppermint oil with PEG 400, or peppermint oilwith Tween® 20, or peppermint oil with sesame oil.

Of course, EC may be substituted or added in place of the HPMC or CMC inthe foregoing examples; PEG 300 or PEG 400 monolaurate can besubstituted or added in place of PEG 400 in the foregoing examples;Tween® 80 may be substituted or added in place of Tween® 20 in theforegoing examples; and other oils such as corn oil, olive oil, soybeanoil, mineral oil or glycerol, may be substituted or added in place ofthe peppermint oil or sesame oil in the foregoing examples.

Further, heating may be applied, for example, heating to a temperatureof about 30° C. to about 90° C., in the course of formulating any ofthese compositions to achieve dissolution of the compounds herein or toobtain an evenly distributed suspension of the NDGA derivative.

In still a further embodiment, the NDGA derivative may be administeredorally as a solid, either without any accompanying carrier or with theuse of carriers. In one embodiment, the NDGA derivative is firstdissolved in a liquid carrier as in the foregoing examples, andsubsequently made into a solid composition for administration as an oralcomposition. For example, the NDGA derivative is dissolved in a modifiedcyclodextrin such as HP-β-CD, and the composition is lyophilized toyield a powder that is suitable for oral administration.

In a further embodiment, the NDGA derivative is dissolved or suspendedin a TPGS solution, with heating as appropriate to obtain an evenlydistributed solution or suspension. Upon cooling, the compositionbecomes creamy and is suitable for oral administration.

In yet another embodiment, the NDGA derivative is dissolved in oil andbeeswax is added to produce a waxy solid composition.

In general, in preparing the oral formulations, the NDGA derivativeherein is first solubilized before other excipients are added so as toproduce compositions of higher stability. Unstable formulations are notdesirable. Unstable liquid formulations frequently form crystallineprecipitates or biphasic solutions. Unstable solid formulationsfrequently appear grainy and clumpy and sometimes contain runny liquids.An optimal solid formulation appears smooth, homogenous, and has a smallmelting temperature range. In general, the proportions of excipients inthe formulation may influence stability. For example, too littlestiffening agent such as beeswax may leave the formulation too runny foran elegant oral formulation.

Hence, in general, for the liquid formulations of the present invention,the excipients used should be good solvents of the NDGA derivativeherein. In other words, the excipients should be able to dissolve theNDGA derivative without heating. The excipients should also becompatible with each other independent of the NDGA derivative such thatthey can form a stable solution, suspension or emulsion. Also, ingeneral, for the solid formulations of the present invention, theexcipients used should also be good solvents of the NDGA derivative toavoid clumps and non-uniform formulations. To avoid solid formulationsthat are too runny or heterogeneous in texture, which are undesirable,the excipients should be compatible with each other such that they forma smooth homogeneous solid, even in the absence of the NDGA derivative.

The present invention further relates to a method of producing thepharmaceutical composition of the present invention, the methodinvolving making or providing the NDGA derivative in a substantiallypurified form, combining the composition with a pharmaceuticallyacceptable carrier or excipient, and formulating the composition in amanner that is compatible with the mode of desired administration.

The compounds and compositions of the present invention find use astherapeutic agents in situations, for example, where one wishes toprovide a treatment to a subject who has a proliferative disease such asa malignant, premalignant or benign tumor, a viral disease, aninflammatory disease, a metabolic disease or a vascular disease.

The compounds and compositions of the present invention can be used totreat a variety of tumors and cancers, including, without limitation,hematological malignancies such as leukemia, for instance acute orchronic lymphoblastic leukemia, acute or chronic myeloid leukemia, acuteor chronic lymphocytic leukemia, acute or chronic myelogenous leukemia,childhood acute leukemia, chronic lymphocytic leukemia, hairy cellleukemia, malignant cutaneous T-cells, mycosis fungoides, non-malignantfibrous cutaneous T-cell lymphoma, lymphomatoid papulosis, T-cell richcutaneous lymphoid hyperplasia, non-Hodgkin's lymphoma, Hodgkin'slymphoma, bullous pemphigoid, discoid lupus erythematosus, lichenplanus, adrenocortical carcinoma, anal cancer, bile duct cancer, bladdercancer, bone cancer, osteosarcoma/malignant fibrous histiocytoma,neurological tumors and malignancies such as neuroblastoma,glioblastoma, astrocytoma, gliomas, brain stem glioma, brain tumorependymoma, medulloblastoma, female and male breast cancer, carcinoidtumor gastrointestinal, carcinoma adrenocortical, carcinoma islet cell,clear cell cancer, clear cell sarcoma of tendon sheaths, colon cancer,colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer,esophageal cancer, Ewing's family of tumors, extragonadal germ celltumor, extrahepatic bile duct cancer, eye cancer, intraocular melanoma,ductal cancer, eye cancer retinoblastoma, dysplastic oral mucosa,invasive oral tumor, gallbladder cancer, gastric (stomach) cancer,gastrointestinal carcinoid tumor, germ cell tumor extragonadal, germcell tumor, gestational trophoblastic tumor, hepatocellular (liver)cancer, hypopharyngeal cancer, intraocular melanoma, islet cellcarcinoma (endocrine pancreas), Kaposi's sarcoma, laryngeal cancer,liver cancer, lung tumors and cancers such as non-small cell lung cancerand small cell lung cancer, malignant mesothelioma, melanoma, merkelcell carcinoma, multiple endocrine neoplasia syndrome, mycosisfungoides, multiple myeloma, nasal cavity tumors, paranasal and sinuscancer, nasopharyngeal cancer, oral cavity and lip cancer, oropharyngealcancer, pancreatic cancer, parathyroid cancer, penile cancer,pheochromocytoma, pineal and supratentorial primitive neuroectodermaltumors, pituitary tumor, pleuropulmonary blastoma, prostate cancer,rectal cancer, renal, pelvis and ureter transitional cell cancer,retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma softtissue adult, Sezary syndrome, skin cancer, small intestine cancer,testicular tumors and cancer, thymoma, thyroid cancer, urethral cancer,transitional and squamous cell urinary carcinoma, gynecological tumorsand cancer such as cervical cancer, ovarian tumors and cancer, ovarianepithelial cancer, ovarian germ cell tumor, uterine cancer, endometrialcancer, vaginal cancer, vulvar cancer, Waldenström's macroglobulinemia,Wilms' tumor, liver tumors including hepatocellular carcinoma (“HCC”)and tumors of the biliary duct, other lung tumors including small celland clear cell cancers, sarcomas in different organs; as well as othercancers and tumors.

Non limiting examples of viral diseases that may be treated effectivelyby the NDGA derivatives of the present invention, include for exampleand without limitation viral infections caused by human immunodeficiencyvirus (HIV), human papillomaviruses (HPV)(all subtypes), herpes simplexvirus 1 and 2 (HSV-1 and HSV-2), Varicella Zoster virus,cytomegalovirus, Epstein Barr virus, pox viruses(smallpox, cowpox,monkeypox, vaccinia), orthohepadnavirus, JC virus, and BK virus, amongothers.

Non-limiting examples of inflammatory diseases that may be treatedeffectively by the NDGA derivatives of the present invention include,for instance and without limitation, rheumatoid arthritis,osteoarthritis, psoriasis, sarcoidosis, systemic lupus erythematosis,Stills disease, cystic fibrosis, chronic obstructive pulmonary diseaseand inflammatory bowel diseases such as ulcerative colitis and Crohns,among others.

Non-limiting examples of metabolic diseases that may be treatedeffectively by the NDGA derivatives of the present invention include,for instance, diabetes mellitus (juvenile onset and adult onset),diabetes insipidis, syndrome X, hyperlipidemia, hypercholesterolemia,hypoglycemia, atheroma, ketoacidosis, Addisons disease, Cushingssyndrome, hyperparathyroidism, hyperthyroidism, leucodystrophy andporphyria, among others.

Non-limiting examples of vascular diseases that may be treatedeffectively by the NDGA derivatives of the present invention include,for instance and without limitation, arterial hypertension, pulmonaryarterial hypertension, cardiovascular disease and macular degeneration,among others.

As mentioned above, an effective amount of the NDGA derivative isadministered to the host, where “effective amount” means a dosagesufficient to produce a desired result. In some embodiments, the desiredresult is at least a reduction in one or more symptoms of the viralinfection or the inflammatory, metabolic or proliferative disease.Typically, the compositions of the present invention will contain fromless than about 0.1% up to about 99% of the active ingredient, that is,the NDGA derivative herein; optionally, the present invention willcontain about 5% to about 90% of the active ingredient. The appropriatedose to be administered depends on the subject to be treated, such asthe general health of the subject, the age of the subject, the state ofthe disease or condition, the weight of the subject, for example.Generally, about 0.1 mg to about 500 mg may be administered to a childand about 0.1 mg to about 5 grams may be administered to an adult. TheNDGA derivative can be administered in a single or, more typically,multiple doses. Preferred dosages for a given agent are readilydeterminable by those of skill in the art by a variety of means in viewof the present disclosure. Other effective dosages can be readilydetermined by one of ordinary skill in the art in view of the presentdisclosure through routine trials establishing dose response curves. Theamount of NDGA derivative will, of course, vary depending upon theparticular NDGA derivative used, as well as the nature of theformulation containing the NDGA derivative, and the route ofadministration.

The frequency of administration of the NDGA derivative, as with thedoses, will be determined by the care giver based on age, weight,disease status, health status and patient responsiveness. Thus, theagents may be administered one or more times daily or as appropriate foras long as needed as conventionally determined.

Kits with multiple or unit doses of the NDGA derivative are included inthe present invention. In such kits, in addition to the containerscontaining the multiple or unit doses of the compositions containing theNDGA derivative will be instructions for its use for a given indicationsuch as an informational sheet or package insert with instructionsdescribing the use and attendant benefits of the drugs in treating thepathological condition of interest, such as any of a number ofinflammatory, metabolic, vascular or proliferative diseases or a viralinfection.

The present invention will now be described in greater detail withreference to the following specific, non-limiting working examples,except as noted where the examples are indicated as being prophetic.

General Procedure.

All reactions were carried out in oven-dried glassware (120° C.) underan atmosphere of nitrogen, unless as indicated otherwise. Acetone,dichloromethane, 1,4-dioxane, ethyl acetate, hexane, and tetrahydrofuranwere purchased Mallinckrodt Chemical Co. Acetone was dried with 4 Åmolecular sieves and distilled. Dichloromethane, ethyl acetate, andhexane were dried and distilled from CaH₂. 1,4-Dioxane andtetrahydrofuran were dried by distillation from sodium and benzophenoneunder an atmosphere of nitrogen. Nordihydroguaiaretic acid was purchasedfrom Fluka Chemical Co. 4-(2-Chloroethyl)morpholine hydrochloride,4-(3-chloropropyl)morpholine hydrochloride, 1-(3-chloropropyl)piperidinemonohydrochloride, 1-(2-chloroethyl)piperidine monohydrochloride,2-chloroethanol, (2-chloroethoxy)ethene, 1-(2-chloroethyl)pyrrolidinehydrochloride, N,N′-dicyclohexylcarbodiimide (DCC),4-dimethylaminopyridine (DMAP), and potassium carbonate were purchasedfrom Aldrich Chemical Co.

The melting point was obtained with a Buchi 535 melting point apparatus.Analytical thin layer chromatography (TLC) was performed on precoatedplates (silica gel 60 F-254), purchased from Merck Inc. Gaschromatographic analyses were performed on a Hewlett-Packard 5890 SeriesII instrument equipped with a 25-m crosslinked methyl silicone gumcapillary column (0.32 mm i.d.). Nitrogen gas was used as a carrier gasand the flow rate was kept constant at 14.0 mL/min. The retention timet_(R) was measured under the following conditions: injector temperature260° C., isothermal column temperature 280° C. Gas chromatography andlow resolution mass spectral analyses were performed on a AgilentTechnology 6890N Network GC System equipped with a Agilent 5973 NetworkMass Selective Detector and capillary HP-1 column. Purification bygravity column chromatography was carried out by use of Merck ReagentsSilica Gel 60 (particle size 0.063-0.200 mm, 70-230 mesh ASTM). Purityof all compounds was >99.5%, as checked by HPLC or GC.

Ultraviolet (UV) spectra were measured on a Hitachi U3300 UV/VISspectrophotometer. Infrared (IR) spectra were measured on a JascoFT-IR-5300 Fourier transform infrared spectrometer. The wave numbersreported were referenced to the polystyrene 1601 cm⁻¹ absorption.Absorption intensities were recorded by the following abbreviations: s,strong; m, medium; w, weak. The fluorescent intensity was measured on aHitach F-4500 Florescence Spectrophotometer. Proton NMR spectra wereobtained on a Varian Mercury-400 (400 MHz) spectrometer by use ofchloroform-d as the solvent and sodium 3-(trimethylsilyl)propionate asinternal standard. Carbon-13 NMR spectra were obtained on a VarianMercury-400 (100 MHz) spectrometer by use of chloroform-d or D₂O as thesolvent. Carbon-13 chemical shifts were referenced to the center of theCDCl₃ triplet (δ77.0 ppm). Multiplicities are recorded by the followingabbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m,multiplet; J, coupling constant (hertz). Hight-resolution mass spectrawere obtained by means of a JEOL JMS-HX110 mass spectrometer.Electrospray ionization mass spectrometry (ESI-MS) analyses wereperformed on a quadrupole ion trap mass analyzer fitted with anelectrospray ionization source of Finnigan LCQ, Finnigan MAT.

Computation was performed on a Silicon Graphics O2+ workstation.Software Chemoffice Ultra 10.0 was used to draw chemical structures andsynthetic schemes. The software PCModel 7.5 was used for energyminimized with the consistent valence force field (CVFF) until themaximum derivative was less than 1.0 kcal mol⁻¹ Å⁻¹.

EXAMPLE 1 Synthesis of 1,4-bis-(3,4-methoxyphenyl) butane (C₂₀H₂₆O₄,FW=330.42) “Compound A”

The compound was synthesized using a modification of Nakamura, et al.²⁰involving coupling of 1,4-diiodobutane with Grignard reagent derivedfrom 3,4-dimethoxybromobenzene in 43% yield. m.p. 93-94° C.(literature²¹ m.p. 91-92° C.). HPLC purity: 99.25%.

¹HNMR (CDCl₃, 300 MHz): δ=1.58-1.68 (m, 4H), 2.55-2.65 (m, 4H), 3.85 (s,6H), 3.86 (s, 6H), 6.69 (s, 2H), 6.70 (dd, J=7.9, 1.6 Hz, 2H), 6.78 (d,J=7.9 Hz, 2H) ppm, consistent with the structure.

¹³CNMR (CDCl₃, 75 MHz): δ=31.1, 35.3, 55.7, 55.9, 111.2, 111.7, 120.1,135.2, 147.0, 148.7 ppm; consistent with the structure.

Analysis: calculated for C₂₀H₂₆O₄, C, 72.70; H, 7.95; found C, 72.51; H:7.82.

MS (EI), m/e=331 (M+1); consistent with C₂₀H₂₆O₄

EXAMPLE 2 Synthesis of 1,4-bis-(3,4-hydroxyphenyl) butane (alternativelynamed 1,4-bis(catechol-4-yl)-butane) [C₁₆H₁₈O₄, FW=274.31) “Compound B”

This compound was synthesized by cleavage of the aromatic methoxy groupof the product of Example 1, (1,4-bis-(3,4-methoxyphenyl) butane), usingboron tribromide²² in quantitative yield. The crude product was found tobe pure by TLC, so it was used for the preparation of derivativeswithout further purification. Analytical sample was purified by a flashsilica gel chromatographic column using dichloromethane and methanol(95:5, V/V) as eluant.

m.p 140-142° C. HPLC purity: 98.5%.

¹H NMR (DMSO-d₆, 300 MHz): δ=1.60 (t, J=6.5 Hz, 4H, 2 CH₂), 2.65 (t,J=6.5 Hz, 4H, 2 CH₂), 6.65-6.85 (m, 6H, 6 Ar—H), 9.56 (brs, 4H, 4OH)ppm; consistent with the structure.

¹³C NMR (DMSO-d₆, 75 MHz): δ=31.5, 36.3, 114.5, 115.8, 120.1, 136.5,143.5, 145.5 ppm, consistent with the structure.

MS (EI), m/e=275 (M+1), consistent with C₁₆H₁₈O₄.

Analysis: calculated for C₁₆H₁₈O₄, C: 70.06, H: 6.61; found, C: 70.31,H, 6.82.

EXAMPLE 3 Synthesis of 1,4-bis-(3,4-ethoxyphenyl) butane (C₂₄H₃₄O₄,FW=386.52) “Compound C”

To a solution of 1,4-bis-(3,4-hydroxyphenyl)butane from Example 2 (700mg, 2.56 mmol) in acetone (26 mL) was added potassium carbonate (2.39 g,16.9 mmol, 6.6 equivalents) and iodoethane (2.40 g, 15.4 mmol, 6.0equivalents) and the mixture heated to reflux for 24 hours. TLC of thereaction indicated completion of the reaction. The reaction mixture wascooled to room temperature, filtered and the solids were washed withacetone. The combined filtrate was concentrated under reduced pressure.The residue was dissolved in ethyl acetate (100 mL) and washed withwater (50 mL). The aqueous layer was re-extracted with ethyl acetate (50mL). The combined organic extracts were dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure to give the crude product (1.06 g),which was crystallized from ethyl acetate-isopropanol (1:1, 6 mL) togive a pure crystalline product (700 mg, 70% yield).

m.p. 113-115° C. HPLC purity: 98.50%.

¹H NMR (CDCl₃, 300 MHz): δ=1.43 (t, J=6.8 Hz, 6H), 1.44 (t, J=6.8 Hz,6H), 1.61-1.65 (m, 4H), 2.40-2.60 (m, 4H), 4.06 (q, J=6.8 Hz, 4H), 4.07(q, J=6.8 Hz, 4H), 6.63-6.72 (m, 4H), 6.79 (d, J=8.2 Hz, 2H) ppm;consistent with the structure.

¹³C NMR (CDCl₃, 75 MHz): δ=14.9, 31.1, 35.3, 64.5, 64.7, 113.9, 114.2,120.4, 135.5, 146.8, 148.6 ppm; consistent with the structure.

Analysis: calculated for C₂₄H₃₄O₄, C: 74.57, H: 8.88; found, C: 74.78,H: 8.75; consistent with the structure.

EXAMPLE 4 Synthesis of1,4-bis{3,4-bis[3-(piperidin-1-yl)propoxy]phenyl}-butane (C₄₈H₇₈N₄O₄,FW=775.16) “Compound D”

This compound could be synthesized by at least two methods.

Method 1: Using Potassium Carbonate as a Catalyst

To a solution of 1,4-bis-(3,4-hydroxyphenyl)butane (1.10 g, 4.0 mmol) inacetone (40 mL) was added potassium carbonate (13.25 g, 96 mmol, 24equivalents), N-(3 chloropropyl)piperidine hydrochloride (9.5 g, 48mmol, 12 equivalents) and sodium iodide (2.4 g, 16 mmol, 4 equivalents)and the mixture was heated to reflux for 40 hours. TLC indicatedcompletion of the reaction. The reaction mixture was filtered and thesolids were washed with acetone. The combined filtrate was concentratedin vacuo. Hexane (200 mL) was added to the residue and the mixture washeated at 60° C. for 15 mm on a Rotavapor™ device. The hexane layer wasdecanted and the procedure was repeated two more times on the residue.The combined hexane extracts were concentrated under reduced pressure togive the crude product. It was purified by silica gel columnchromatography using silica gel (250 g) and gradient elution withhexane: ethyl acetate: triethylamine (5:5:0.5) to ethylacetate:methanol:triethylamine (9:1:0.5) to give the pure product (1.56g, 50.4% yield) as a white solid. It was further purified bycrystallization from ethyl acetate-hexane to give a crystalline product(1.03 g).

m.p. 91-93° C. HPLC purity: 99.43%.

¹H NMR (CDCl₃, 100 MHz): δ=1.38-1.50 (m, 8H), 1.53-1.68 (m, 20H),1.95-2.07 (m, 8H), 2.35-2.60 (m, 28H), 3.9 (t, J=6.4 Hz, 4H), 4.0 (t,J=6.4 Hz, 4H), 6.63-6.72 (m, 4H), 6.77 (d, J=8.0 Hz, 2H) ppm, consistentwith the structure.

¹³C NMR (CDCl₃, 75 MHz): δ=24.5, 27.0, 31.3, 35.4, 54.7, 56.2, 67.9,68.1, 114.4, 114.7, 120.6, 135.7, 147.1, 149.0 ppm, consistent with thestructure.

Analysis: calculated for C₄₈H₇₈N₄O₄, C: 74.37, H: 10.16, N: 7.23; found,C: 74.07, H: 10.06, N: 7.13.

MS (ESI): m/z=803.7 (M+H)⁺, 802.7 (M), 678.6, 402.4, 126.0, consistentwith the structure.

Method 2: Using Sodium Hydride as a Catalyst

To a solution of 1,4-bis(3,4-dihydroxyphenyl)-butane (13.0 g, 45.78mmols) in anhydrous DMF (1 L) was added 60% suspension of sodium hydridein paraffin (9.9 g, 248 mmol 5.4 equivalents) and the mixture was heatedat 65° C. for one hour. Then the mixture was cooled to room temperature,N-(3-chloropropyl)piperidine (37.0 g, 229 mmol 5.0 equivalents) andsodium iodide (6.9 g, 46 mmol 1 equivalent) were added and the mixturewas allowed to stir at room temperature for 120 hours. TLC indicatedcomplete conversion to the product.

The workup of the reaction was carried out by slow addition of thereaction mixture to water (3 L) and diethyl ether (2.5 L). The aqueouslayer was extracted again with diethyl ether (2 L). The combined organicextracts were washed with brine (750 mL), dried over anhydrous sodiumsulfate and concentrated under reduced pressure. The crude product waspurified by silica gel column chromatography. The column was built usingsilica gel (500 g) and solvent mixture ethylacetate:methanol:triethylamine (93:2:5) and eluted with the gradientethyl acetate:methanol:triethylamine (93:2:5 to 91:4:5) to give theproduct as white solid (26.7 g, yield 75.4%). This product wascrystallized from ethyl acetate: hexane mixture to give crystallinecompound (21.4 g).

Analytical data are identical to those obtained from sample prepared bymethod 1.

EXAMPLE 5 Synthesis of1,4-bis{3,4-bis[3-(piperidin-1-yl)propoxy]phenyl}-butanetetrakis-hydrochloride salt (C₄₈H₇₈N₄O₄.4HCl, FW=921.00) “Compound E”

To all ice cooled (0-5° C.) solution of aqueous concentrated HCl (7.0mL, of 11 N, 77 mmol, 24 equiv.) in 95% ethanol (21 mL) was addeddropwise a solution of1,4-bis{3,4-bis[3-(piperidin-1-yl)propoxy]phenyl}-butane (2.50 g, 3.225mmol) in 95% ethanol (21 mL)*. The solution was allowed to stir at 0-5°C. for three hours and the solvent was removed on a rotary evaporatorkeeping the temperature of the water bath at 45° C. The hydrochloridesalt was dried under high vacuum for 48 hours. The crude product wasthen crystallized from ethanol: ether to give 2.46 g of the product(83.2% yield) after drying high vacuum for 72 hours. *Note that thestarting material1,4-bis{3,4-bis[3-(piperidin-1-yl)propoxy]phenyl}-butane did notdissolve completely in 95% ethanol at room temperature, so the mixturewas heated at 45° C. during which1,4-bis{3,4-bis[3-(piperidin-1-yl)propoxy]phenyl}-butane dissolved. Thesolution was cooled to room temperature and added to the ethanolic HClsolution.

The analytical data for this product are given below.

m.p. 270-280° C. (dec.). HPLC purity: 98.5%. Moisture content by KarlFisher method: 2.4974%.

Elemental Analysis: C₄₈H₈₂N₄O₄Cl₄, required, C: 62.59, H: 8.97, and N:6.08; found, C: 62.38, H: 9.30; and N: 5.89.

Chlorine elemental analysis by titration method (anhydrous basis):theory, 15.40%; found: 15.44% (100.3% of the theory).

¹H NMR (D₂O, 300 MHz): δ=1.25-1.50 (m, 8H), 1.55-1.75 (m, 12H), 1.82 (d,J=14.4 Hz, 8H), 2.06-2.10 (m, 8H), 2.39 (s, 4H), 2.76 (t, J=12.1 Hz,8H), 3.11 (q, J=7.8 Hz, 8H), 3.39 (d, J=11.6 Hz, 8H), 3.98-4.00 (m, 8H),6.64 (dd, J=1.8, 8.0 Hz, 2H), 6.79 (d, J=1.8 Hz, 2M), 6.84 (d, J=8.0,2H) ppm; consistent with the structure.

¹³C NMR (D₂O, 100 MHz): δ=24.5, 27.0, 31.3, 35.4, 54.7, 56.2, 67.9,68.1, 114.4, 114.7, 120.6, 135.7, 147.1, 149.0 ppm; consistent with thestructure.

MS (ESI), m/z=777 (M⁺+2), 775 (M⁺); consistent with the free baseC₄₈H₇₈N₄O₄ (775.16).

EXAMPLE 6 Synthesis of1,4-bis{3,4-bis[2-(1-methyl-piperazin-4-yl)-ethoxy]phenyl}-butane(C₄₄H₇₄N₈O₄; FW=779.11) “Compound F”

Step 1. Synthesis of N′-methyl-N-2-chloroethylpiperazine from itsdihydrochloride salt

The N′-methyl-N-2-chloroethylpiperazine dihydrochloride (10.0 g, 45.4mmol) was added slowly to a mixture of 50% aqueous potassium carbonate(200 mL) and ether (200 mL) and the mixture was stirred for 30 min.,layers were separated, aqueous layer extracted with ether (2×200 mL).Combined organic extracts were dried on anhydrous sodium sulfate andconcentrated to give N′-methyl-N-2-chloroethylpiperazine (6.2 g).

Step 2: synthesis of1,4-bis{3,4-bis[2-(1-methyl-piperazin-4-yl)-ethoxy]phenyl}-butane

To a solution of compound 2 (1.0 g, 3.65 mmol) in acetone (40 mL) wereadded anhydrous potassium carbonate (2.52 g, 18.25 mmol, 5 equivalents),and N′-methyl-N-2-chloroethylpiparazine (2.90, 18.25 mmol) were addedand the mixture was refluxed. After about 36 hours, additional amountsof potassium carbonate (2.528 g) and N′-methyl-N-2-chloroethylpiparazine(2.90 g) were added and refluxing was continued for 36 more hours. Thereaction mixture was cooled to room temperature, filtered, washed withacetone (200 mL) and concentrated. The crude was purified by silica gel(250 g) column chromatography using gradient elution withCH₂Cl₂:MeOH:Et₃N respectively of 85:10:5 to 65:30:5, to give the titlecompound (0.88 g, 31.1%). It was further purified by crystallizationfrom ethyl acetate-hexane.

HPLC purity: 99.67%.

¹H NMR (CDCl₃, 300 MHz): δ=1.55-1.63 (m, 4H), 2.28 (s, 6H), 2.29 (s,6H), 2.33-2.63 (m, 36H), 2.81 (t, J=6.0 Hz, 4H), 2.82 (t, J=6.0 Hz, 4H),4.10 (q, J=6.0 Hz, 8H), 6.67 (dd, J=8.2 Hz and 1.7 Hz, 2H), 6.70 (d,J=1.7 Hz, 2H), 6.78 (d, J=8.2 Hz, 2H) ppm; consistent with thestructure.

¹³C NMR (CDCl₃, 75 MHz): δ=31.2, 35.3, 46.1, 53.7, 55.1, 57.3, 67.3,67.4, 114.4, 114.7, 120.9, 146.0, 147.9, 148.7 ppm; consistent with thestructure.

Analysis: calculated for C₄₄H₇₄N₈O₄, C: 67.83, H: 9.59 and N: 14.39;found, C: 67.53, H: 9.71 and N: 14.03.

EXAMPLE 7 Synthesis of1,4-bis{3,4-bis(2-methyl-thiazol-4-yl-methoxy)phenyl}-butanetetrakis-hydrochloride salt (C₃₆H₃₈N₄O₄S₄, FW=718.97) “Compound G”

First, 4-chloromethyl-2-methyl thiazole was generated from itshydrochloride. This was done by adding 4-chloromethyl-2-methyl thiazolehydrochloride (2.5 g, 13.5 mmol) to 50% aqueous potassium carbonate andether (30 mL each). The mixture was stirred for 15 minutes. The organiclayer was separated, dried over anhydrous K₂CO₃ and concentrated to give4-chloromethyl-2-methyl thiazole (2.01 g). This was used in thesynthesis of1,4-bis{3,4-bis(2-methyl-thiazol-4-yl-methoxy)phenyl}-butanetetrakishydrochloride salt as follows.

To an ice cooled solution of 1,4-bis-(3,4-hydroxyphenyl)butane fromExample 2 (616 mg, 2.25 mmol) in DMF (15 mL) was added a 60% suspensionof sodium hydride in paraffin (54 1 mg, 13.5 mmol, 6 equivalents). Themixture was stirred at 0° C. for 30 minutes and at room temperature for30 minutes. Then a solution of the 4-chloromethyl-2-methyl thiazole(2.01 g, 13.5 mmol, 6 equivalents) in DMF (5 mL) was added and thereaction mixture was allowed to stir for 16 hours at room temperature.The reaction mixture was added to the saturated aqueous NH₄Cl (150 mL)and ether (350 mL). After shaking, the organic layer separated, and theaqueous layer was extracted with ether (150 mL). The combined organicextracts were washed with water (50 mL), brine (50 mL), and dried overanhydrous Na₂SO₄ and concentrated under reduced pressure to give thecrude product. It was purified by silica gel column chromatography usingsilica gel (250 g) and hexane:ethyl acetate (50:50 to 0:100) as aneluant to give the product as a white solid (0.88 g, 54.3%). It wasfurther purified by crystallization form ethyl acetate-hexane.

m.p. 96-98° C. HPLC purity: 99.99%.

¹H NMR (CDCl₃) 1.51-1.61 (m, 4H), 2.48-2.60 (m, 4H), 2.71 (s, 6H), 2.72(s, 6H), 5.20 (s, 4H), 5.22 (s, 4H), 6.69 (dd, J=8.2 Hz, 1.8, 2H), 6.80(d, J 1.8 Hz, 2H), 6.88 (d, J=8.2 Hz, 2H), 7.18 (s, 4H) ppm; consistentwith the structure.

¹³C NMR (CDCl₃) 19.1, 30.9, 35.2, 67.8, 68.0, 115.3, 115.4, 115.6,115.7, 121.5, 136.4, 146.8, 148.5, 152.4, 152.5, 166.1 ppm; consistentwith the structure.

Analysis: calculated for C₃₆H₃₈N₄O₄S₄, C: 60.13, H: 5.34, and N: 7.79.Found C 60.07, H 5.22, and N: 7.70.

EXAMPLE 8 Synthesis of1,4-bis{3,4-bis(2-(N,N′-dimethylamino)-ethoxy)phenyl}t-butane(C₃₂H₅₄N₄O₄, FW=558.41) “Compound H”

To a solution of 1,4-bis(3,4-dihydroxyphenyl)-butane (1.096 g, 4 mmol)in acetone (40 mL) were added anhydrous potassium carbonate (6.64 g, 48mmol) and dimethylaminoethyl chloride hydrochloride (3.46 g, 24 mmol)and the mixture was heated to reflux. After 12 hours an additionalamount of potassium carbonate (6.648 g) and dimethylaminoethylchloridehydrochloride (3.46 g) were added and the reaction mixture was heated toreflux for 64 hours. TLC of the reaction mixture indicated completion.The reaction mixture was cooled to room temperature, filtered, washedwith acetone (150 mL) and concentrated. The crude obtained was purifiedby silica gel (250 g) column chromatography using gradient elutionCH₂Cl₂:MeOH:Et₃N respectively of 94:1:5 to 85:10:5 to give the product(1.28 g, 57.8%). This material was crystallized from ethyl acetatehexaneto give further purified product (300 mg).

m. p. 65-67° C. HPLC purity: 99.04%

¹H NMR (CDCl₃, 300 MHz): δ=1.59-1.63 (m, 4H), 2.33 (s, 12H), 2.34 (s,12H), 2.50-2.56 (m, 4H), 2.73 (t, J=6.1 Hz, 4H), 2.74 (m, J=6.1 Hz, 4H),4.07 (q, J=6.1 Hz, 8H), 6.68 (dd, J=7.9 Hz, 1.9 Hz, 2H), 6.7 1 (d, J=1.9Hz, 2H), 6.80 (d, J=7.9 Hz, 2H) ppm; consistent with the structure.

¹³C NMR (CDCl₃, 75 MHz): δ=31.1, 35.3, 45.9, 46.1, 58.3, 67.8, 67.191, 40.6, 114.9, 120.9, 136.0, 147.1, 148.9 ppm, consistent with thestructure.

Analysis: calculated for C₃₂H₅₄O₄N₄, C: 68.78, H: 9.76, and N: 10.03;found, C: 69.82, H: 9.85, and N: 9.83.

As illustrated in the following prophetic Examples A-R, additionalchemical reactions can be performed to synthesize compounds according toembodiments of the present invention.

Prophetic Example A Alternative synthesis of 1,4-bis-3,4-hydroxyphenyl)butane (alternatively named 1,4-bis(catechol-4-yl)-butane)” in Example 2“Compound B”

Prophetic Example B Synthesis of1,4-bis{3,4-bis[2-(piperidin-1-yl)ethoxy]phenyl}-butane-tetrakis-hydrochloridesalt; free base (C₄₄H₇₀N₄O₄, FW=719.05); 4HCl salt (C₄₄H₇₀N₄O₄.4HCl,FW=864.89)

Prophetic Example C Synthesis of1,4-bis{3,4-bis[3-(morpholin-1-yl)propoxy]phenyl}-butanetetrakis-hydrochloride salt free base (C₄₄H₇₀N₄O₈, FW=783.05); 4HCl salt(C₄₄H₇₀N₄O₈.4HCl, FW=928.89)

Prophetic Example D Synthesis of1,4-bis{3,4-bis[2-(morpholin-1-yl)ethoxy]phenyl}-butanetetrakis-hydrochloride salt free base (C₄₀H₆₂N₄O₈, FW=726.94); 4HCl salt(C₄₀H₆₂N₄O₈.4HCl, FW=872.79)

Prophetic Example E Synthesis of1,4-bis{3,4-bis[2-(pyrrolidin-1-yl)ethoxy]phenyl}-butanetetrakis-hydrochloride salt free base (C₄₀H₆₂N₄O₄, FW=662.94); 4HCl salt(C₄₀H₆₂N₄O₄.4HCl, FW=808.79)

Prophetic Example F Synthesis of1,4-bis{3,4-bis[2-(1-methyl-piperazin-4-yl)-ethoxy]phenyl}-butanetetrakishydrochloride salt free base (C₄₄H₇₄N₈O₄; FW=779.11); 4HCl salt:(C₄₄H₇₄N₈O₄.4HCl, FW=924.95)

Prophetic Example G Synthesis of1,4-bis{3,4-bis(2-hydroxyethoxy)phenyl}-butane free base (C₂₄H₃₄O₈,FW=450.23)

Method 1

Method 2

Method 3

Prophetic Example H Synthesis of1,4-bis{3,4-bis(2-(N,N-di(2-hydroxyethyl)amino-ethoxyl)phenyl}-butanetetrakis-hydrochloride salt free base (C₄₀H₇₀N₄O₁₂, FW=799.00); 4HClsalt (C₄₀H₇₀N₄O₁₂.4HCl, FW=944.85)

Prophetic Example I Synthesis of1,4-bis{3,4-bis[2-(2-hydroxyethoxy)ethoxyl]phenyl}-butane free base(C₃₂H₅₀O₁₂, FW=626.73)

Prophetic Example J Synthesis of1,4-bis{3,4-bis[2-(piperidin-1-yl)ethylcarbamoyloxy]phenyl}-butanetetrakis-hydrochloride salt free base (C₄₈H₇₄N₈O₈, FW=890.15), 4HCl salt(C₄₈H₇₄N₈O₈.4HCl, FW=1036.99)

Method 1

Method 2

Prophetic Example K Synthesis of1,4-bis{3,4-bis[2-(morpholin-1-yl)ethylcarbamoyloxy]phenyl}-butanetetrakis-hydrochloride salt free base (C₄₄H₆₆N₈O₁₂, FW=899.04); 4HClsalt (C₄₄H₆₆N₈O₁₂.4HCl, FW=1044.89)

Prophetic Example L Synthesis of1,4-bis{3,4-bis[(2-N,N-dimethylaminoethyl)carbamoyloxy]phenyl}-butanefree base (C₃₆H₅₈N₈O₈, FW=730.89)

Prophetic Example M Synthesis of1,4-bis{3,4-bis[(furan-2-yl)methyl-carbamoyloxy]phenyl}-butane free base(C₄₀H₃₈N₄O₁₂, FW=766.75)

Prophetic Example N Synthesis of1,4-bis(3,4-dimethoxyphenyl)-2,3-bis(chloromethyl)-(2S,3R)-butane(C₂₂H₂₈Cl₂O₄, FW=427.36)

Prophetic Example O Synthesis of1,4-bis(3,4-dimethoxyphenyl)-2,3-bis(bromomethyl)-(2S,3R)-butane(C₂₂H₂₈Br₂O₄, FW=516.26)

Prophetic Example P Synthesis of1,4-bis(3,4-dimethoxyphenyl)-2,3-bis(fluoromethyl)-(2S,3R)-butane(C₂₂H₂₈F₂O₄, FW=394.45)

Prophetic Example Q Synthesis of1,4-bis(3,4-dimethoxyphenyl)-2,3-diformyl-(2S,3R)-butane (C₂₂H₂₆O₆,FW=386.44)

Prophetic Example R Synthesis of1,4-bis(3,4-dimethoxyphenyl)-2,3-dicyano-(2S,3R)-butane (C₂₂H₂₄N₂O₄,FW=380.44)

The invention will further be described with respect to the followingspecific, non-limiting working examples relating to in vitrocytotoxicity and effectiveness studies, following the general protocolsthat were performed.

Cytotoxicity and Effectiveness Studies

Certain of the compounds of the present invention have been studied invitro. The in vitro studies have established that the various classes ofthe NDGA derivatives of the present invention would be safe andeffective for prophylactic or after-onset treatment of a viral infectionor a proliferative, inflammatory, metabolic or vascular disease. Thefollowing examples explain the studies involved in such testing.

Cytotoxicity Studies

Studies performed regarding cytotoxicity included the well-known MTS,Trypan Blue and MTT protocols. The MTS studies were done using theCellTiter 96®AQ_(ueous) One Solution Cell Proliferation Assay (PromegaCorporation, Madison, Wis. USA). Metabolically active, namely viable,cells turn MTS, a tetrazolium compound(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt) into colored formazan, which is soluble in tissue culturemedium. The measurement of the absorbance of the formazan is read at 490nm. The ready to use reagent is added directly to the cells in media in96-well plates, incubated 1-4 hours and the results are recorded by theplate reader. The IC₅₀ is estimated by graphing the gathered data. TheIC₅₀ is the concentration of the tested material that inhibits 50% ofgrowth or viability of the tested material compared to a control.

In the Trypan Blue assay, cells are trypsinized and a sample is added toa solution of trypan blue dye and saline. Viable cells are able to keepthe dye on the outside of their membrane, damaged or dead cells are not.Viable and non-viable (blue) cells are counted, and the percent viableis calculated. The proliferation rate is calculated using the valuesfrom placebo treated cells and compares the viability of treated cellsto that of non-treated cells.

The MTT assay is a colorimetric method for determining the number ofviable cells. Metabolically active cells turn the MTT reagent,(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, intopurple-colored formazan crystals that are solubilized in dimethylsulfoxide (DMSO). The MTT reagent is added to MTT (colorless) media. Themedia is added to the cells and they are incubated for 4 hours. DMSO isadded to the cells and mixed. The colored solutions are then read at 540nm and corrected at 630 nm. The IC₅₀ is estimated by graphing thegathered data.

Antiviral Activity SEAP Assay

Antiviral activity is determined using a SEAP (SEcreted AlkalinePhosphatase) assay, in which cells are co-transfected with SEAP and TATplasmids. TAT is a transactivator of human immunodeficiency virus (HIV)gene expression and is one of the two or more necessary viral regulatoryfactors (TAT and REV) for HIV gene expression. TAT acts by binding tothe TAR RNA element and activating transcription from the long terminalrepeat (LTR) promoter. The TAT protein stabilizes elongation oftranscription and has also been shown to be involved in transcriptioninitiation. Previous studies have shown that TAT mediates reduction ofantibody-dependent T cell proliferation, contributing substantially tothe failure of the immune response. TAT also directly stimulatesKaposi's cell growth.

Since TAT has no apparent cellular homologs, this strong positiveregulator has become an attractive target for the development ofanti-AIDS drugs. In contrast to currently available HIV reversetranscriptase inhibitors (AZT, DDI) or potential protease inhibitorsthat prevent new rounds of infection, an inhibitor which suppressesviral gene TAT regulated expression of integrated proviral DNA willarrest the virus at an early stage (Hsu et al., Science 254:1799-1802,1991). Efforts aimed at the elucidation of factors which control geneexpression at transcriptional and post-transcriptional levels in hosteukaryotes have recently made possible quantitative assessment of TATfunction (Sim, Ann. N.Y. Acad. Sci. 616: 64-70, 1990). To screen forinhibitors for TAT regulated transactivation (TAT-TRS), the SEAPreporter gene is put under the control of HIV-1 LTR promoter in theplasmid pBC12/HIV/SEAP. The TAT-coded activity is supplied by a secondplasmid construct pBC12/CMV/t2. Transient co-transfection of COS-7 cellswith these two plasmids leads to secretion of alkaline phosphatase intothe culture medium which is analyzed by a simple colorimetric assay(Berger et al., Gene 66: 1-10, 1988). The SEAP assay, therefore,provides an indirect determination of TAT transactivation.

In the SEAP assay, an inhibitor should cause reduction of SEAP reportergene expression via transactivation of the HIV-1 LTR promoter by TATprotein (TAT-TRS). The TAT protein is expressed under the control of acytomegalovirus promoter and induces the expression of thenon-endogenous, heat resistant form of secreted alkaline phosphatase. IfTAT transactivation is blocked by a drug, the reporter SEAP will not beexcreted into the media. SEAP is detected colorimetrically by thepara-nitrophenyl phosphate substrate at 405 nm. See Gnabre¹³. The cellsare analyzed by the MTT assay to compare cytotoxicity to SEAPinhibition. The goal is for the drug to inhibit SEAP without being tootoxic.

Antiprolifeative Activity

Antiproliferative activity is determined using the TiterTACS® ApoptosisDetection Kit (R&D Systems Inc., Minneapolis, Minn. USA) relating toapoptosis of cells based on DNA fragmentation, and by the ELISA VEGF(vascular epithelial growth factor) and survivin assays.

In the DNA fragmentation assay, in situ detection of apoptosis isspecifically achieved with TiterTACS® 96-well Apoptosis Detection kit,Catalog No. TA600) (R&D Systems, Inc.), The TiterTACS® assay providesquantification of apoptosis in cultured cells without direct counting oflabeled cells using colorimetric detection. Cells are treated with thetest compounds, left untreated as experimental negative controls, ortreated with TACS nuclease as positive controls. TACS nuclease allowspositive controls to be generated for each experimental system: a brieftreatment of cells with the TACS nuclease prior to labeling generatesDNA breaks in every cell, providing an appropriate positive controlspecific for the system under study. The TdT enzyme that catalyzes theaddition of dNTPs to DNA fragments allows for colorimetric detection ofat 450 nm by using a streptavidin-HRP solution followed by theTACS-Sapphire substrate. A high absorbance at 450 nm is indicative ofapoptosis in the cells. Treated cells are compared to untreated cellsand to nuclease-cleaved cells to assess the extent of apoptosis.

ELISA (Enzyme-Linked ImmunoSorbent Assay) VEGF studies were donefollowing manufacturer's instructions using the Endogen® Human VEGFELISA kit, Catalog No. EHVEGF (Pierce Biotechnology, Inc., Rockford,Ill. USA). Hypoxic conditions are created for the cells by treating themwith desferrioxamine (DFO), which chelates iron and causes the cells tosecrete VEGF into the media. The supernatant (media) was removed andfrozen for testing, and the cells were counted with the Trypan Blueassay so that the results can be normalized to cell count. The VEGF ismeasured with a sandwich ELISA that captures the protein in media on anantibody-coated microplate, and then uses a biotinylated antibodyreagent to detect the protein. Results from a standard curve enable theuser to quantify the amount of protein in each sample.

ELISA-Survivin Assay

Survivin is an inhibitor of apoptosis that is abundantly expressed inmany human cancers, but not in normal adult human tissue, and isconsidered a possible modulator of the terminal effector phase of celldeath/survival. Survivin is expressed in G₂-M in a cell cycle-dependentmanner, binding directly to mitotic spindle microtubules. It appearsthat survivin phosphorylation on Thr34 may be required to maintain cellviability at cell division, and expression of aphosphorylation-defective survivin mutant has been shown to triggerapoptosis in several human melanoma cell lines. Phosphorylated survivinacts on the caspase pathway to suppress the formation of caspase-3 andcaspase-9, thereby inhibiting apoptosis. Thus, compounds that reduce theexpression of survivin will be expected to increase the rate ofapoptosis and cell death.

Effects of the tested compounds on survivin were studied followingmanufacturer's instructions using the Surveyor™ IC Human Total SurvivinImmunoassay, Catalog No. SUV647 (R&D Systems, Inc.). Cell lysates wereanalyzed for survivin protein content. The kit includes a plate coatedwith an antibody specific for survivin and a biotinylated antibodyreagent that recognizes survivin bound to the plate. The plate is readon a plate reader set at 450 nm and corrected at 540 nm. A protein assayaccording to the Bradford method (Bradford, M. 1976, Anal Biochem 72:248-254) was used to quantify and normalize the samples according tototal protein content. Results from a standard curve enable the user toquantify the amount of survivin protein in each sample.

Anti-Inflammatory Activity

Anti-inflammatory activity was determined based on the effect of testedcompounds on primary human keratinocytes (PHKs). PHKs play an importantrole in inflammatory processes, synthesizing a number of cytokines,adhesion molecules and growth factors. Studies were conducted todetermine whether tested compounds could inhibit keratinocytes toprevent or reduce production of interferon gamma (IFN-γ), interleukin-8(IL-8), tumor necrosis factor alpha (TNF-α), granulocyte/macrophagecolony-stimulating factor (GM-CSF), intercellular adhesion molecule—1(ICAM-1, also known as CD54) and monocyte chemotactic protein-1 (MCP-1).PHKs are first treated with TNF-α to induce a pro-inflammatory state andrelease of pro-inflammatory cytokines. Specific cytokines are thenassayed following manufacturer's instructions using R&D Systems, Inc.'sprotein assays (Quantikine ELISA kits; Catalog Nos. DCP00 for MCP-1 kit,DGM00 for GM-CSF kit, and DIF50 for IFN-γ).

General Protocols for Cytotoxicity Studies Relating to Antiviral andAntiproliferative Activity

Three cell lines obtained from ATCC and maintained as directed by ATCCwere tested: HeLa (cervical adenocarcinoma), A549 (lung carcinoma), andCOS-7 (SV40 transformed monkey kidney). Studies of these cell lines areconsidered to be indicative of the effect of tested substances onmammalian, including human, diseases.

All compounds were dissolved in DMSO and DMSO is used as the placebo.The samples were first dissolved into 10 mM dilutions in DMSO and thendiluted further to 5, 1, 0.5, 0.1, 0.05, and 0.01 mM solutions. Thesesolutions were added to the media at a 1% concentration (1 μl to 100 μlmedia) to treat the cells with 100, 50, 10, 5, 1, 0.5, and 0.1 μM of thecompound. The compounds that were found to have low IC₅₀s were dilutedfurther in DMSO. Solutions were checked before use for precipitation,especially the 10 mM solutions. If there was precipitation, they werewarmed 65° C. and added to warmed media.

Wells on a 96-well plate were seeded with 1.5-8×10⁴ cells in 100 μlmedia and incubated overnight. The outside wells of the plate werefilled with 200 μl sterile, deionized (DI) water to curb mediaevaporation. After 24 hours, test chemicals were prepared in media at aconcentration of 1% media volume. The test sample media were added,mixing with the pipette before adding 100 μl per well. Media was addedto the “Media Only” wells. The well plate and its contents wereincubated for 24-72 hours, depending on the study conducted. On the dayof the MTS assay, the MTS reagent was removed from the refrigerator andbrought to room temperature. Using the multichannel pipette, 20 μl ofthe MTS reagent was added to each well and incubated for one to fourhours. The plate was read at 490 nm with a reference wavelength of 690nm after one hour in the incubator thereafter until the blank wells wereat about 0.2 OD. The results were then scanned into a Microsoft® Excel®template designed to perform all necessary calculations when data areentered. The data were checked for statistical errors; data points thatare within 10% of the mean of the data points for that group wereincluded. The average of the blanks (“Media Only”) were subtracted andthe data were inserted into a chart that represents growth response,treated/untreated.

SEAP Protocol

The following SEAP protocol was used as a reporter system for measuringthe activity of TAT-mediated transactivation of HIV transcription. TheMTT assay measures cellular proliferation and is used to verify that thelevels of SEAP activity are not solely due to cytotoxicity. These assayswas used in screening for potential drug compound leads as antiviralagents, and particularly, anti-HIV candidates.

COS-7 (green monkey kidney) cells were co-transfected using Fugene 6reagent (Roche Applied Science, Cat. No. 11815091001, Indianapolis,Ind., USA) with two plasmids: pHIVSEAP (SEAP expression vector under thecontrol of the HIV LTR promoter) and pCTAT (HIV TAT transcription factorexpression vector under the control of a CMV (cytomegalovirus)promoter). After test compound treatment for 48 hours, samples wereanalyzed for SEAP activity at 405 nm after addition of the p-nitrophenylphosphate substrate. The percentage of SEAP activity inhibition wascalculated in relation to the placebo control.

EXAMPLE 9 SEAP/MTT and MTS Studies—Cytotoxicity and AntiviralEffectiveness

The results of the SEAP/MTT and MTS studies on the indicated compoundsfrom the working Examples above based on the general protocols set forthabove are set forth in the following Table 3, where the left hand columnidentifies the compound tested and EC₅₀ indicates the concentration ofthe compound having 50% of the effect of the control. IC₅₀ was definedabove.

TABLE 3 SEAP COS-7 Compound EC₅₀ IC₅₀ A549 IC₅₀ HeLa IC₅₀ A >100 μM >100μM >100 μM >100 μM C >100 μM >100 μM >100 μM >100 μM D 0.7-0.8 μM0.8-0.9 μM 1-2 μM 5-6 μM E 9 μM 5-10 μM F >100 μM >100 μM G >100 μM >100μM H 30-40 μM 20-30 μM 20-40 μM 20-30 μM

Results from compounds with biological activity in two cell lines testedusing the MTS assay as an indicator of cell viability are shown in Table3. IC₅₀ concentrations show various degrees of inhibition of cellviability by these compounds. All compounds were able to reduce cellviability, and therefore, induce apoptosis, in a dose dependent manner.Compound D showed the strongest cell viability inhibition in A549 andHeLa cells, while its hydrochloride salt (Compound E) showed the secondstrongest inhibition in these two cell lines.

The extent of inhibition of TAT-transactivation as determined by SEAPassay also varied among compounds, as also shown in Table 3. Compound Dalso showed significant anti-viral activity with EC₅₀ around 0.7-0.8 μM.Inhibitory concentrations of cell viability in the test cells (COS-7) asdetermined by MTT were usually similar as the effective concentrationsfor TAT-transactivation.

EXAMPLE 10 Antiproliferative Activity Based on TiterTACS®, VEGA andSurvivin Aptoptosis Studies

The results of the TiterTACS® DNA fragmentation, ELISA VEGA and ELISAsurvivin apoptosis studies on the indicated compounds from the workingExamples above, based on the general protocols above, are set forth inthe following Table 4.

TABLE 4 DNA Survivin Compound fragmentation IC₅₀ VEGF IC₅₀ D Positive 4μM H Positive 25 μM >60 μM Positive = induces apoptosis Negative = doesnot induce apoptosis

The compounds tested have shown significant reduction of survivinprotein levels at low micromolar concentrations. Consequently, they havealso shown strong apoptosis induction as determined by the DNAfragmentation assay. Compound H reduces survivin protein expression andis therefore able to induce apoptosis. Compound D is also an inducer ofapoptosis. These compounds are able to decrease survivin and induceapoptosis in a dose-dependent manner. Compound H was the most potentinhibitor of survivin protein production with IC₅₀ for survivinproduction around 25 μM.

Release of VEGF protein is also decreased by these compounds. Inhibitionof VEGF protein production ranged in the low micromolar concentrationsfor compound D Inhibition by all compounds was observed in adose-dependent fashion. Compound D showed an IC₅₀ for VEGF productionaround 4 μM, while compound H showed VEGF IC₅₀ at higher than 60 μM.

The results from Table 4 indicate that the compounds of the presentinvention are effective in inhibiting viral activity and causing celldeath (apoptosis) in the cell lines tested. Of the compounds tested,Compounds D, E, and H appear to be more effective than the others due totheir lower EC₅₀ and IC₅₀ values. Compound D appears to be the mosteffective.

EXAMPLE 11 U.S. National Cancer Institute DTP Human Tumor Cell LineScreen

The United States National Cancer Institute (NCI) provides adevelopmental therapeutics program (DTP)(http://dtp.nci.nih.gov/branches/btb/ivclsp.html) to screen submittedsubstances in support of cancer drug discovery. The In Vitro Cell LineScreening Project (IVCLSP) is a dedicated service providing directsupport to the DTP anticancer drug discovery program and is designed toscreen up to 3,000 compounds per year for potential anticancer activity.The operation of this screen utilizes 60 different human tumor celllines, representing leukemia, melanoma and cancers of the lung, colon,brain, ovary, breast, prostate, and kidney. The aim is to prioritize forfurther evaluation, synthetic compounds or natural product samplesshowing selective growth inhibition or cell killing of particular tumorcell lines. This screen is unique in that the complexity of a 60 cellline dose response produced by a given compound results in a biologicalresponse pattern which can be utilized in pattern recognition algorithms(COMPARE program. See:http://dtp.nci.nih.gov/docs/compare/compare.html). Using thesealgorithms, it is possible to assign a putative mechanism of action to atest compound, or to determine that the response pattern is unique andnot similar to that of any of the standard prototype compounds includedin the NCI database. In addition, following characterization of variouscellular molecular targets in the 60 cell lines, it may be possible toselect compounds most likely to interact with a specific moleculartarget.

The screening is a two-stage process, beginning with the evaluation ofall compounds against the 60 cell lines at a single dose of 10 μM. Theoutput from the single dose screen is reported as a mean graph and isavailable for analysis by the COMPARE program. Compounds which exhibitsignificant growth inhibition are evaluated against the 60 cell panel atfive concentration levels.

Methodology of the In Vitro Cancer Screen

The human tumor cell lines of the cancer screening panel are grown inRPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine.For a typical screening experiment, cells are inoculated into 96 wellmicrotiter plates in 100 μL at plating densities ranging from 5,000 to40,000 cells/well depending on the doubling time of individual celllines. After cell inoculation, the microtiter plates are incubated at37° C., 5% CO₂, 95% air and 100% relative humidity for 24 hours prior toaddition of experimental drugs.

After 24 hours, two plates of each cell line are fixed in situ withtrichloroacetic acid (TCA), to represent a measurement of the cellpopulation for each cell line at the time of drug addition (Tz).Experimental drugs are solubilized in DMSO at 400-fold the desired finalmaximum test concentration and stored frozen prior to use. At the timeof drug addition, an aliquot of frozen concentrate is thawed and dilutedto twice the desired final maximum test concentration with completemedium containing 50 μg/ml gentamicin. Additional four, 10-fold or ½ logserial dilutions are made to provide a total of five drug concentrationsplus control. Aliquots of 100 μl of these different drug dilutions areadded to the appropriate microtiter wells already containing 100 μl ofmedium, resulting in the required final drug concentrations.

Following drug addition, the plates are incubated for an additional 48hours at 37° C., 5% CO₂, 95% air, and 100% relative humidity. Foradherent cells, the assay is terminated by the addition of cold TCA.Cells are fixed in situ by the gentle addition of 50 μl of cold 50%(w/v) TCA (final concentration, 10% TCA) and incubated for 60 minutes at4° C. The supernatant is discarded, and the plates are washed five timeswith tap water and air dried. Sulforhodamine B (SRB) solution (100 μl)at 0.4% (w/v) in 1% acetic acid is added to each well, and plates areincubated for 10 minutes at room temperature. After staining, unbounddye is removed by washing five times with 1% acetic acid and the platesare air dried. Bound stain is subsequently solubilized with 10 mM trizmabase, and the absorbance is read on an automated plate reader at awavelength of 515 nm. For suspension cells, the methodology is the sameexcept that the assay is terminated by fixing settled cells at thebottom of the wells by gently adding 50 μl of 80% TCA (finalconcentration, 16% TCA). Using the seven absorbance measurements [timezero, (Tz), control growth, (C), and test growth in the presence of drugat the five concentration levels (Ti)], the percentage growth iscalculated at each of the drug concentrations levels. Percentage growthinhibition is calculated as:[(Ti−Tz)/(C−Tz)]×100 for concentrations for which Ti>/=Tz[(Ti−Tz)/Tz]×100 for concentrations for which Ti<Tz.

Three dose response parameters are calculated for each experimentalagent. Growth inhibition of 50% (GI₅₀) is calculated from[(Ti−Tz)/(C−Tz)]×100=50, which is the drug concentration resulting in a50% reduction in the net protein increase (as measured by SRB staining)in control cells during the drug incubation. The drug concentrationresulting in total growth inhibition (TGI) is calculated from Ti=Tz. TheLC₅₀ (concentration of drug resulting in a 50% reduction in the measuredprotein at the end of the drug treatment as compared to that at thebeginning) indicating a net loss of cells following treatment iscalculated from [(Ti−Tz)/Tz]×100=−50. Values are calculated for each ofthese three parameters if the level of activity is reached; however, ifthe effect is not reached or is exceeded, the value for that parameteris expressed as greater or less than the maximum or minimumconcentration tested.

A summary of the results of the NCI DTP study as described above forCompound A at 10 μM is set forth in Table 5:

TABLE 5 Compound D Cell Line Panel Mean Growth (# cell lines) Percent at10 μM GI₅₀ TGI LC₅₀ NSCLC (9) 62.77 3.39E−6 7.31E−6 2.84E−5 Colon Cancer(6) 40.49 2.36E−6 6.45E−6 2.28E−5 Breast Cancer (8) 59.72 1.00E−52.11E−5 3.08E−5 Ovarian Cancer (6) 66.18 6.23E−6 1.46E−5 3.53E−5Leukemia (6) 12.31 1.09E−6 4.28E−6 1.34E−5 Renal Cancer (8) 82.548.00E−6 1.70E−5 3.70E−5 Melanoma (8) 16.46 4.09E−6 7.74E−6 1.59E−5Prostate (1) 2.84 1.41E−5 2.76E−5 5.39E−5 CNS (5) 78.88 1.90E−6 4.13E−61.10E−5

The results indicate that Compound D at 10 μM concentration hadbroad-based activity in reducing cell growth against a number of thetypes of the 60 cell lines. The strongest effects in cell growthreduction were shown against the prostate cancer cell lines, followed byleukemia cell lines, with the least drastic effects shown against renalcancer. GI₅₀, TGI and LC₅₀ values were all lowest in leukemia and CNScancer cell lines, indicating the highest potency of Compound D againstthose cancer cell types.

EXAMPLE 12

Anti-Inflammatory and Anti-Vascular Disease Activity PHK Studies

Anti-inflammatory activity of several of the compounds of the previouslydescribed working examples was determined based on the effect of testedcompounds on primary human keratinocytes (PHKs) as described above andfollowing the manufacturer's instructions for the various kits used inthe studies. While these studies more typically are used mostly foranti-inflammatory investigations, they can also apply to vasculardiseases, since the results in keratinocytes may be extrapolated tovascular endothelium.

Only MCP-1 data with PHKs is dose-dependent, so an IC₅₀ for MCP-1 can becalculated, but not for the other cytokines Results of the MCP-1 studieson various compounds are shown in the following Table 6.

TABLE 6 MCP-1 Compound IC₅₀ A >100 μM C >100 μM D 3 μM H 75 μM

Results from compounds with anti-inflammatory activity in TNF-α-treatedPHK as assayed by MCP-1 protein production are shown in Table 6. IC₅₀concentrations for MCP-1 protein show various degrees of inhibition ofthis inflammatory cytokine. In results of other of these tests, allcompounds that were tested were able to reduce MCP-1 protein productionin a dose dependent manner. Compound D showed the strongest inhibitionof MCP-1 in TNF-α-treated PHK.

EXAMPLE 13 Permeability Studies as Indicative of Oral Bioavailability

Permeability studies were conducted by an outside contractor on behalfof the assignee of the present invention and application to determinethe permeability of Compounds D and G through Caco-2 monolayers. Animportant factor of oral bioavailability is the ability of a compound tobe absorbed in the small intestine. Measurement of drug apparentpermeability (P_(app)) through cell monolayers is well correlated withhuman intestinal absorption, and several mammalian cell lines, includingCaco-2, LLC-PK1 and MDCK, are appropriate for this measurement(Artursson, P. et al., “Correlation between oral drug absorption inhumans and apparent drug permeability coefficients in human intestinalepithelial (Caco-2) cells,” Biochem Biophys Res Comm 175:880-885 (1991);Stewart, B. H., et al., “Comparison of intestinal permeabilities inmultiple in vitro and in situ models: relationship to absorption inhumans,” Pharm Res 12:693 (1995)). P-Glycoprotein (P-gp, encoded byMDR1) is a member of the ABC transporter super family and is expressedin the human intestine, liver and other tissues. Intestinal expressionof P-gp may affect the oral bioavailability of drug molecules that aresubstrates for this transporter. Interaction with P-gp can be studiedusing direct assays of drug transport in polarized cell systems such asCaco-2 cell monolayers, and human P-gp cDNA-expressing LLC-PKi and MDCKcell monolayers.

Caco-2 cells (human adenocarcinoma colonic cell line Caco-2, ATCC Cat.No. HTB-37, used between passages 18 and 45) were seeded onto BD Falcon™HTS 24-Multiwell, 1 μm culture inserts (BD Catalog No. 351180) (BDBiosciences Discovery Labware, Woburn, Mass., USA). The cells werecultured for 21-25 days with media replacement every 3-4 days. Monolayerintegrity was evaluated by pre-experimental trans-epithelial electricalresistance (TEER) measurements and post-experimental lucifer yellow A toB flux determinations.

Transport buffer was HBSS (Hanks Balanced Salt solution) buffered withthe addition of 10 mM HEPES(N-[2-Hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]), and pHadjusted to 7.4 with NaOH. Receiver solution was prepared by adding 1%DMSO to transport buffer. The test solution was of Compounds D and H inDMSO in transport buffer at a final DMSO concentration of 1%. Donorsolutions for two permeability comparator compounds (50 μM[³H]-propranolol as “High” and 50 μM [¹⁴C]-mannitol as “Low”) as well asa positive control P-gp substrate (5 μM [³H]-digoxin) were prepared bydiluting aliquots of radiolabeled and non-radiolabeled stock solutionsinto transport buffer at a final DMSO concentration of 1%.

The test articles Compounds D and H were assayed at a singleconcentration (1 μM) in both A to B and B to A directions. The donor andreceiver solutions were added to the apical or basolateral chambers ofthe monolayers (depending on the direction of transport to be measured).The monolayers were incubated on an orbital shaker (50 rpm) at 37° C.,with ambient humidity and CO₂. After 90 minutes, samples from the donorand receiver chambers were removed for analysis.

To determine the extent of non-specific binding of the sample to theassay plate, the sample solution was incubated under the conditionsdescribed above in a single well of a 24-well assay plate. After 90minutes, the sample was removed from each well for analysis. After allsamples were collected, lucifer yellow solution was added to eachmonolayer at a final concentration of 100 μM. The inserts were placed ina new receiver plate containing transport buffer. After a 30 minincubation on an orbital shaker (50 rpm) at 37° C., with ambienthumidity and CO₂, samples were removed from the receiver chamber tomeasure percent lucifer yellow flux.

Two permeability comparator compounds representing high permeability (50μM [³H]-propranolol) and low permeability (50 μM [¹⁴C]-mannitol) wereassayed in the A to B direction, with samples from the donor andreceiver chambers taken at one time point of 90 minutes. The controlP-gp substrate was 5 μM [³H]-digoxin assayed in both A to B and B to Adirections, with samples from the donor and receiver chambers taken atone time point of 90 minutes. Duplicate monolayers were used for eachincubation.

Samples were analyzed by LC/MS/MS using peak area ratios to an internalstandard. Test article concentrations were calculated based on the peakarea ratios obtained for appropriate dilutions of the donor solutioncontaining the test article at the nominal concentration in transportbuffer. Samples were diluted as appropriate to ensure that the responsewas within the linear range of the mass spec detector. Comparator andcontrol samples were analyzed by liquid scintillation counting. Luciferyellow concentrations were determined using a fluorescence plate reader.

Pre-experimental trans-epithelial electrical resistance (TEER)measurements (mean 411 Ωcm²) and post-experimental lucifer yellow A to Bflux (mean 0.1%) confirmed monolayer integrity. The polarization of thepositive control digoxin confirmed a functioning P-gp transport model.The P_(app) values for the permeability comparators mannitol andpropranolol were within historical ranges observed at the testingfacility with this test system (mean 4.1×10⁻⁷±SD 2.4×10⁻⁷ for mannitol,1.5×10⁻⁵±4.2×10⁻⁵ for propranolol) indicating a properly functioningmodel.

The results for the permeability comparators mannitol and propranolol aswell as for the control P-gp substrate digoxin are reported in thefollowing Table 7.

TABLE 7 P_(app) Polarization Mass [cm/s] Ratio balance mean mean meanCompound A to B B to A (B-A/A-B) A to B B to A digoxin (5 μM) 2.28E−061.24E−05 5.4 73% 79% propranolol 1.42E−05 n.d. n.d. 71% n.d. (50 μM)mannitol (50 μM) 5.14E−07 n.d. n.d. 96% n.d. n.d.—not determined

Bidirectional permeability data as well as polarization ratios obtainedfor the test compounds in Caco-2 cell monolayers are reported in Table8.

TABLE 8 Nominal Polarization conc. P_(app) [cm/s] Ratio Test article[μM] A to B B to A [B-A/A-B] Compound D 1.0 1.38E−05 1.40E−05 2.35E−062.51E−06 0.18 Compound H 20 6.67E−05 6.44E−05 1.01E−05 1.01E−05 0.15

The recovery of the test articles from the apical and basolateralchambers (mass balance) as well as from a single well of a 24-wellculture plate without cells (non-specific binding) at the end of theincubation is reported in Table 9.

TABLE 9 Non- Nominal specific Test conc. Mass Balance * Binding article[μM] A to B B to A % Recovery Com- 1.0    54%    78% 48%    39% 35%pound D Com- 20 ≧100% ≧100% 90% ≧100% 96% pound H * Massbalance/recovery values of >100% are typically due to increasedsolubility over the course of the incubation

Conclusions

The results were very encouraging. Permeability studies in Caco-2 cellmonolayer as an in vitro model of intestinal absorption demonstrate thatCompound H falls under the high permeability class, and that Compound Dfalls under the moderate-to-high permeability class as determined by theBCS permeability classification system.

Monolayer TEER results as well as post-experimental lucifer yellow fluxresults indicated the presence of functional cell monolayers throughoutthe assay. The positive control for P-gp transport, digoxin, showedpolarized transport (polarization ratio of 5.4, Table 7) indicating aproperly functioning test system.

Recovery of Compound H from the apical and basolateral chambers at theend of the assay (mass balance) as well as from the assay plate withoutcells (Table 9) was complete, indicating that the degree of non-specificbinding of Compound H to the cells or to the plastic plate did notaffect the assay. However, mass balance and non-specific bindingdeterminations showed that Compound D was bound to the plastic plateand/or the cells to a degree that might have reduced the amount ofcompound available for diffusion and transport. As a result, theapparent P_(app) values and resulting polarization ratios (Table 8) mayunderestimate the permeability of Compound D and should be used withcaution when assigning the compounds to a BCS permeability class asshown in Table 10.

TABLE 10 Absorption in Permeability Class^([3]) Humans^([4]) Papp(cm/sec) low <50 % ≦mannitol moderate 50-89 % >mannitol and <propranololhigh ≧90 % ≧propranolol

The A-B permeability observed for Compound H (Table 8) in Caco-2 cellmonolayers was higher than that of propranolol. Based on the BCSpermeability classification (Table 10), Compound H should therefore beconsidered a high permeability compound. The A-B permeability valuesobserved for Compound D were between those observed for mannitol andpropranolol. Based on the BCS permeability classification (Table 10),Compound D would be considered moderate-to-high permeability compounds.As noted above, however, these classifications should be interpretedwith caution in light of the degree of binding observed for thesecompounds.

Compounds D and H showed polarization ratios of 0.1-0.2, indicating thatA-B permeability exceeded B-A permeability for these compounds. Thisfinding is inconsistent with P-gp mediated B-A efflux, and may suggestinvolvement of active A-B flux by a transporter other than P-gp.

The foregoing studies demonstrate that representative compounds of theNDGA derivatives of the present invention would be useful pharmaceuticalcompounds.

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications within the spirit and scope of thepresent invention as defined by the appended claims.

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We claim:
 1. A butane bridge modified nordihydroguaiaretic acidderivative compound (BB-N) having the following general structure(Formula V), and its pharmaceutically acceptable salts:

wherein each X is selected from the group consisting of —CN, —CH2Cl,—CH2Br and —CH2F.
 2. A composition comprising the BB-N compound of claim1 and a pharmaceutically acceptable carrier, optionally with otherpharmaceutically acceptable excipients.
 3. A method of treating adisease selected from breast cancer, lung cancer, skin cancer,esophageal cancer, prostate cancer, glioma or leukemia comprisingadministering to a subject an amount of the BB-N compound of claim 1alone or as part of a pharmaceutical composition.
 4. A method oftreating breast cancer, lung cancer, skin cancer comprisingadministering to a subject an amount of the BB-N compound of claim 1alone or as part of a pharmaceutical composition.
 5. A method oftreating lung cancer comprising administering to a subject an amount ofthe BB-N compound of claim 1 alone or as part of a pharmaceuticalcomposition.
 6. A method of treating skin cancer comprisingadministering to a subject an amount of the BB-N compound of claim 1alone or as part of a pharmaceutical composition.
 7. A kit comprising apharmaceutical composition comprising the BB-N compound of claim 1 andinstructions for its use in relieving, alleviating or amelioratingbreast, lung, skin, esophageal, prostate cancer, glioma or leukemia. 8.The pharmaceutically acceptable salt of claim 1 which is selected fromthe following acids HCl, HBr, HNO3, MeSO3H, H2SO4, aspartic acid, citricacid, benzenesulfonic acid, camphoric acid, camphorsulfonic acid,ethanesulfonic acid, ethansulfonic acid, formic acid, fumaric acid,galactaric acid, D-gluconic acid, glycolic acid, hippuric acid, L-lacticacid, maleic acid, malic acid, malonic acid, nicotinic acid, palmiticacid, pamoic acid, phosphoric acid, salicylic acid, succinic acid,tartaric acid, p-toluenesulfonic acid.
 9. A method oftreating-esophageal cancer comprising administering to a subject anamount of the BB-N compound of claim 1 alone or as part of apharmaceutical composition.
 10. A method of treating prostate cancercomprising administering to a subject an amount of the BB-N compound ofclaim 1 alone or as part of a pharmaceutical composition.
 11. A methodof treating glioma comprising administering to a subject an amount ofthe BB-N compound of claim 1 alone or as part of a pharmaceuticalcomposition.
 12. A method of treating leukemia comprising administeringto a subject an amount of the BB-N compound of claim 1 alone or as partof a pharmaceutical composition.